Particles comprising a therapeutic or diagnostic agent and suspensions and methods of use thereof

ABSTRACT

The invention provides particles, compositions including the particles, and methods of making the particles using electrospray. In certain embodiments, the particles allow for high concentrations of a therapeutic or diagnostic agent to be delivered at low viscosity. Particles may also exhibit beneficial properties with respect to stability.

BACKGROUND OF THE INVENTION

There is an urgent and unmet need for therapeutic and diagnosticformulations which are reliable, convenient, and cost-effective toadminister. This is particularly true in relation to therapeuticproteins, e.g., monoclonal antibodies, which are increasingly importantin the treatment of a wide range of life-threatening and debilitatingdiseases. Desirable formulations comprise a high concentration oftherapeutic or diagnostic agents and confer appropriate stability, suchthat a minimal volume of the formulation can be used to administer ahigh dose of the agents. In some cases this helps to curtail the time todeliver the required dose and the pain or discomfort experienced by thepatient. In some cases, this may also help to decrease the frequency ofadministration of the agents. Such formulation attributes are, however,typically unattainable in aqueous solution. High concentrations ofaqueous therapeutic or diagnostic agents are often typified by highfluid dynamic viscosity, precluding the use of standard injectiondevices, while degradation of the active ingredient proceeds through oneor several pathways at an accelerated rate.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of forming particles byelectrospraying, e.g., conventional electrospraying, a stream of a firstliquid including a first therapeutic or diagnostic agent toward acollector (e.g., another liquid), the particles being collected on thecollector, the concentration of the first therapeutic or diagnosticagent in the liquid ranging from 0.0001 to 1000 mg/mL, e.g., 1 to 1000mg/mL, 1 to 900 mg/mL, 1 to 500 mg/mL, 1 to 250 mg/mL, 1 to 100 mg/mL, 1to 50 mg/mL, 5 to 1000 mg/mL, 100 to 900 mg/mL, 150 to 800 mg/mL, or 200to 700 mg/mL, and the viscosity of the liquid ranging from 0.1 to 5000cP, e.g., 0.75 to 1.5 cP, 0.1 to 1000 cP, 0.1 to 100 cP, 1 to 5000 cP,10 to 1000 cP, or 100 to 500 cP. In some embodiments, the inventionprovides a method of forming particles by electrospraying an annularstream of an encapsulant in a second liquid toward the collector, andcentrally with respect to the annular steam of encapsulant,electrospraying a stream of the first liquid. In some embodiments, anencapsulant is in the first liquid.

In another aspect, the invention provides a method of electrospraying,e.g., conventional electrospraying, a first liquid including a firsttherapeutic or diagnostic agent to form droplets and removing (e.g.,evaporating) the first liquid to produce particles from the droplets.The therapeutic or diagnostic agent in the particles has 0.5 to 1.0activity per unit, e.g., 0.75 to 1.0 activity per unit, or 0.9 to 1.0activity per unit.

In either method, the method may further include suspending theparticles in a pharmaceutically acceptable medium, thereby forming apharmaceutical composition. Alternatively, the particles may beformulated as a pharmaceutical composition in dry form, e.g., a powder.

In some embodiments, the encapsulant includes poly(vinyl alcohol),poly(acrylic acid), poly(acrylamide), poly(ethylene oxide), poly(lacticacid), poly(glycolic acid), polycaprolactone, poly(lactic-co-glycolicacid), chitosan, cellulose, or any combination thereof. In someembodiments, the encapsulant is any excipient, therapeutic agent, ordiagnostic agent.

In other embodiments, the first liquid is aqueous, an organic solvent,an ionic liquid, a hydrogel, an ionogel, or a combination thereof.Organic solvents include benzyl alcohol, benzyl benzoate, castor oil,coconut oil, corn oil, cottonseed oil, fish oil, grape seed oil,hazelnut oil, hydrogenated palm seed oil, olive oil, peanut oil,peppermint oil, safflower oil, sesame oil, soybean oil, sunflower oil,vegetable oil, walnut oil, polyethylene glycol, glycofurol, acetone,diglyme, dimethylacetamide, dimethyl isosorbide, dimethyl sulfoxide,ethanol, ethyl acetate, ethyl ether, ethyl lactate, isopropyl acetate,methyl acetate, methyl isobutyl ketone, methyl tert-butyl ether,N-methyl pyrrolidone, perfluorodecalin, 2-pyrrolidone, trigylcerides,tetrahydrofurfuryl alcohol, triglycerides of the fractionated plantfatty acids C8 and C10 (e.g., MIGLYOL® 810 and MIGLOYL® 812N), propyleneglycol diesters of saturated plant fatty acids C8 and C10 (e.g.,MIGLYOL® 840), ethyl oleate, ethyl caprate, dibutyl adipate, fatty acidesters, hexanoic acid, octanoic acid, triacetin, diethyl glycolmonoether, gamma-butyrolactone, eugenol, clove bud oil, citral,limonene, and any combination thereof. Aqueous liquids include water,0.9% saline, lactated Ringer's solution, and buffers (e.g., acetatebuffer, histidine buffer, succinate buffer, HEPES buffer, tris buffer,carbonate buffer, citrate buffer, phosphate buffer, glycine buffer,barbital buffer, and cacodylate buffer). The liquid may further includeanother component, such as a carbohydrate, a pH adjusting agent, a salt,a chelator, a mineral, a polymer, a surfactant, a protein stabilizer, anemulsifier, an antiseptic, an amino acid, an antioxidant, a protein, anorganic solvent, or nutrient media. In some embodiments, each of theother components is, independently, at 0.0001 to 99% (w/v) of sprayedliquid, e.g., at 0.0001 to 90% (w/v), at 0.0001 to 50% (w/v), at 0.0001to 10% (w/v), at 0.0001 to 1% (w/v), or at 0.0001 to 0.1% (w/v). One ofordinary skill in the art would be able to determine an appropriateamount of the other components in the sprayed liquid. Carbohydratesinclude dextran, trehalose, sucrose, agarose, mannitol, lactose,sorbitol, and maltose. The pH adjusting agent may be, e.g., acetate,citrate, glutamate, glycinate, histidine, lactate, maleate, phosphate,succinate, tartrate, bicarbonate, aluminum hydroxide, phosphoric acid,hydrochloric acid, DL-lactic/glycolic acids, phosphorylethanolamine,tromethamine, imidazole, glyclyglycine, or monosodium glutamate. Saltsinclude sodium chloride, calcium chloride, potassium chloride, sodiumhydroxide, stannous chloride, magnesium sulfate, sodium glucoheptonate,sodium pertechnetate, or guanidine hydrochloride. The chelator can be,e.g., disodium edetate. The mineral can be, e.g., calcium, zinc, ortitanium dioxide. Suitable polymers include propyleneglycol, glucosestar polymer, silicone polymer, polydimethylsiloxane, polyethyleneglycol, carboxymethylcellulose, poly(glycolic acid),poly(lactic-co-glycolic acid), and polylactic acid. The surfactant canbe, e.g., polysorbate, magnesium stearate, sodium dodecyl sulfate,polyethylene glycol nonylphenyl ether (Triton™ N-101), glycerin, orpolyoxyethylated castor oil. Protein stabilizers includeacetyltryptophanate, caprylate, and N-acetyltryptophan. The emulsifiercan be, e.g., polysorbate 80, polysorbate 20, sorbitan monooleate,ethanolamine, polyoxyl 35 castor oil, poloxyl 40 hydrogenated castoroil, carbomer 1342, a corn oil-mono-di-triglyceride, a polyoxyethylatedoleic glyceride, or a poloxamer. Antiseptics include phenol, m-cresol,benzyl alcohol, 2-phenyloxyethanol, chlorobutanol, neomycin,benzethonium chloride, gluteraldehyde, or beta-propiolactone. The aminoacid may be, e.g., alanine, aspartic acid, cysteine, isoleucine,glutamic acid, leucine, methionine, phenylalanine, pyrrolysine, serine,selenocysteine, threonine, tryptophan, tyrosine, valine, asparagine,L-arginine, histidine, glycine, or glutamine, e.g., asparagine,L-arginine, histidine, glycine, or glutamine. The antioxidant can be,e.g., glutathione, ascorbic acid, cysteine, or tocopherol. The proteincan be, e.g., protamine, protamine sulfate, or gelatin. The organicsolvent can be dimethyl sulfoxide or N-methyl-pyrrolidone,N-ethyl-pyrrolidone, or a mixture thereof. Suitable preservativesinclude methyl hydroxybenzoate, thimerosal, parabens, formaldehyde, andcastor oil. The liquid may further include adenine, tri-n-butylphosphate, octa-fluoropropane, white petrolatum, orp-aminophenyl-p-anisate. Exemplary ionic liquids may contain pyridinium,pyridazinium, pyrimidinium, pyrazinium, imidazolium, pyrazolium,thiazolium, oxazolium, triazolium, ammonium, sulfonium, halides,sulfates, sulfonates, carbonates, phosphates, bicarbonates, nitrates,acetates, PF₆—, BF₄—, triflate, nonaflate, bis(trifyl)amide,trifluoroacetate, heptafluorobutanoate, haloaluminate, or anycombination thereof. Exemplary hydrogels or ionogels are collagenhydrogels, chitosan hydrogels, methylcellulose hydrogels, dextranhydrogels, alginate hydrogels, agarose hydrogels, poly(methylmethacrylate) hydrogels, poly(amido amine) hydrogels,poly(ethyleneimine) hydrogels, polyethylene oxide hydrogels, gelatinhydrogels, hyaluronic acid hydrogels, and any combinations thereof.

In some embodiments, the pharmaceutical composition has a concentrationof the first therapeutic or diagnostic agent from 0.0001 to 1000 mg/mL,e.g., 100 to 800, 200 to 700, 200 to 600, or 300 to 700 mg/mL.

In some embodiments, the pharmaceutical composition includes a secondtherapeutic or diagnostic agent, e.g., at a concentration from 0.0001 to1000 mg/mL. The first and second therapeutic or diagnostic agents can bethe same or different.

Therapeutic and diagnostic agents include nucleic acids,oligonucleotides, antibodies, amino acids, peptides, proteins, cells,bacteria, gene therapeutics, genome engineering therapeutics, epigenomeengineering therapeutics, carbohydrates, chemical drugs, contrastagents, magnetic particles, polymer beads, metal nanoparticles, metalmicroparticles, quantum dots, antioxidants, antibiotic agents, hormones,nucleoproteins, polysaccharides, glycoproteins, lipoproteins, steroids,analgesics, local anesthetics, anti-inflammatory agents, anti-microbialagents, chemotherapeutic agents, exosomes, outer membrane vesicles,vaccines, viruses, bacteriophages, adjuvants, vitamins, minerals,organelles, and any combination thereof.

In some embodiments, any of a second liquid being electrosprayed, aliquid collector, or suspension medium is aqueous, an organic solvent,an ionic liquid, a hydrogel, ionogel, or a combination thereof. Organicsolvents for use in the medium include benzyl alcohol, benzyl benzoate,castor oil, coconut oil, corn oil, cottonseed oil, fish oil, grape seedoil, hazelnut oil, hydrogenated palm seed oil, olive oil, peanut oil,peppermint oil, safflower oil, sesame oil, soybean oil, sunflower oil,vegetable oil, walnut oil, polyethylene glycol, glycofurol, acetone,diglyme, dimethylacetamide, dimethyl isosorbide, dimethyl sulfoxide,ethanol, ethyl acetate, ethyl ether, ethyl lactate, isopropyl acetate,methyl acetate, methyl isobutyl ketone, methyl tert-butyl ether,N-methyl pyrrolidone, perfluorodecalin, 2-pyrrolidone, trigylcerides,tetrahydrofurfuryl alcohol, triglycerides of the fractionated plantfatty acids C8 and C10 (e.g., MIGLYOL® 810 and MIGLOYL® 812N), propyleneglycol diesters of saturated plant fatty acids C8 and C10 (e.g.,MIGLYOL® 840), ethyl oleate, ethyl caprate, dibutyl adipate, fatty acidesters, hexanoic acid, octanoic acid, triacetin, diethyl glycolmonoether, gamma-butyrolactone, eugenol, clove bud oil, citral,limonene, and any combination thereof. Exemplary aqueous liquids arewater, 0.9% saline, lactated Ringer's solution, and buffers (e.g.,acetate buffer, histidine buffer, succinate buffer, HEPES buffer, trisbuffer, carbonate buffer, citrate buffer, phosphate buffer, glycinebuffer, barbital buffer, and cacodylate buffer). The medium may furtherinclude another component, such as a carbohydrate, a pH adjusting agent,a salt, a chelator, a mineral, a polymer, a surfactant, a proteinstabilizer, an emulsifier, an antiseptic, an amino acid, an antioxidant,a protein, an organic solvent, or nutrient media. In some embodiments,each of the other components is, independently, at 0.0001 to 99% (w/v)of the liquid, e.g., at 0.0001 to 90% (w/v), at 0.0001 to 50% (w/v), at0.0001 to 10% (w/v), at 0.0001 to 1% (w/v), or at 0.0001 to 0.1% (w/v).One of ordinary skill in the art would be able to determine anappropriate amount of the other components in the liquid. Carbohydratesinclude dextran, trehalose, sucrose, agarose, mannitol, lactose,sorbitol, or maltose. pH adjusting agents include acetate, citrate,glutamate, glycinate, histidine, lactate, maleate, phosphate, succinate,tartrate, bicarbonate, aluminum hydroxide, phosphoric acid, hydrochloricacid, DL-lactic/glycolic acids, phosphorylethanolamine, tromethamine,imidazole, glyclyglycine, or monosodium glutamate. Salts include sodiumchloride, calcium chloride, potassium chloride, sodium hydroxide,stannous chloride, magnesium sulfate, sodium glucoheptonate, sodiumpertechnetate, or guanidine hydrochloride. An exemplary chelator isdisodium edetate. Minerals include calcium, zinc, and titanium dioxide.Polymers include propyleneglycol, glucose star polymer, siliconepolymer, polydimethylsiloxane, polyethylene glycol,carboxymethylcellulose, poly(glycolic acid), poly(lactic-co-glycolicacid), and polylactic acid. Surfactants include polysorbate, magnesiumstearate, sodium dodecyl sulfate, polyethylene glycol nonylphenyl ether(Triton™ N-101), glycerin, or polyoxyethylated castor oil. Proteinstabilizers include acetyltryptophanate, caprylate, orN-acetyltryptophan. The emulsifier can be, e.g., polysorbate 80,polysorbate 20, sorbitan monooleate, ethanolamine, polyoxyl 35 castoroil, poloxyl 40 hydrogenated castor oil, carbomer 1342, a cornoil-mono-di-triglyceride, a polyoxyethylated oleic glyceride, or apoloxamer. Antiseptics include phenol, m-cresol, benzyl alcohol,2-phenyloxyethanol, chlorobutanol, neomycin, benzethonium chloride,gluteraldehyde, and beta-propiolactone. The amino acid may be, e.g.,alanine, aspartic acid, cysteine, isoleucine, glutamic acid, leucine,methionine, phenylalanine, pyrrolysine, serine, selenocysteine,threonine, tryptophan, tyrosine, valine, asparagine, L-arginine,histidine, glycine, or glutamine, e.g., asparagine, L-arginine,histidine, glycine, or glutamine. Suitable antioxidants includeglutathione, ascorbic acid, cysteine, and tocopherol. The protein can beprotamine, protamine sulfate, or gelatin. The organic solvent can bedimethyl sulfoxide, N-ethyl-pyrrolidone, N-methyl-pyrrolidone, ormixtures thereof. The preservative can be, e.g., methyl hydroxybenzoate,thimerosal, parabens, formaldehyde, or castor oil. The medium mayfurther include, e.g., adenine, tri-n-butyl phosphate,octa-fluoropropane, white petrolatum, or p-aminophenyl-p-anisate. Ionicliquids may contain, e.g., pyridinium, pyridazinium, pyrimidinium,pyrazinium, imidazolium, pyrazolium, thiazolium, oxazolium, triazolium,ammonium, sulfonium, halides, sulfates, sulfonates, carbonates,phosphates, bicarbonates, nitrates, acetates, PF₆—, BF₄—, triflate,nonaflate, bis(trifyl)amide, trifluoroacetate, heptafluorobutanoate,haloaluminate, or any combination thereof. Exemplary hydrogels orionogels arecollagen hydrogels, chitosan hydrogels, methylcellulosehydrogels, dextran hydrogels, alginate hydrogels, agarose hydrogels,poly(methyl methacrylate) hydrogels, poly(amido amine) hydrogels,poly(ethyleneimine) hydrogels, polyethylene oxide hydrogels, gelatinhydrogels, hyaluronic acid hydrogels, and any combinations thereof.

In certain embodiments, the amount of additional compound, i.e.,excipient, present in the first liquid, second, liquid, collectingliquid, or medium, is as shown in the following table. The percentagesare as a percentage of the total solute loading by weight. For a firstliquid with a therapeutic at a concentration of 10 mg/mL and anexcipient at a concentration of 5 mg/mL, e.g., the weight fraction ofthe excipient, relative to the total solute population, is 33%.

Excipient Range 1 Range 2 Range 3 Range 4 Carbo-   10-30%    3-50%   1-80%   0.3-99% hydrate pH adusting  0.5-5%   0.2-40%  0.05-70%  0.01-99% agent Salt   10-50%    3-70%    1-85%   0.3-99% Chelator 0.01-1%  0.003-40%  0.001-80%  0.0003-99% Mineral   10-50%    3-70%   1-80%   0.3-99% Polymer   10-60%    3-75%    1-85%   0.3-99%Surfactant  .01-1%  0.003-40%  0.001-80%  0.0003-99% Protein   10-70%   3-70%    1-85%   0.3-99% stabilizer Emulsifier  .01-1%  0.003-40% 0.001-80%  0.0003-99% Antiseptic   .5-10%   0.2-50%  0.05-70%  0.02-99% Amino acids   10-25%    3-50%    1-85%   0.3-99% Antioxidant 0.01-1%  0.003-40%  0.001-80%  0.0003-99% Protein    1-10%   0.3-50%  0.1-75%   0.03-99% Organic 0.001-2% 0.0003-1% 0.0001-10% 0.00003-99%solvent Nutrient   10-50%    3-70%    1-85%   0.3-99% mediaFor second liquids, collecting liquids, and medium, Range 4 may be to100% for any excipient listed in the table.

In some embodiments, the particles have diameters from 0.1 to 1000 μm,e.g., 1 to 400 μm, 1 to 200 μm, 1 to 100 μm, 1 to 50 μm, 1 to 25 μm, 1to 10 μm, 10 to 100 μm, 50 to 100 μm, 50 to 75 μm, or 75 to 100 μm.

In certain embodiments, the particles have a polydispersity index from0.05 to 0.9.

In some embodiments, the pharmaceutical composition has a viscosity from0.27 to 200 cP, e.g., 0.27 to 100 cP, 0.27 to 50 cP, 0.27 to 30 cP, 20to 50 cP, 1 to 30 cP, 1 to 20 cP, or 1 to 15 cP.

In certain embodiments, the pharmaceutical composition includes from 5to 90% particles by volume, e.g., 20 to 90%, 40 to 80%, 50 to 60%, or 70to 90%.

In a related aspect, the invention provides a composition includingparticles made by a method of the invention.

In another aspect, the invention provides a method of administering afirst therapeutic or diagnostic agent by administering a pharmaceuticalcomposition including particles made by a method of the invention.

In another aspect, the invention provides a method of administering afirst therapeutic or diagnostic agent to a mammal. The method includesadministering an effective amount of a pharmaceutical composition to themammal. In certain embodiments, the pharmaceutical composition includesa medium and particles including the therapeutic or diagnostic agent,where the pharmaceutical composition has a viscosity from 0.27 to 200cP, e.g., 0.27 to 100 cP, 0.27 to 50 cP, 0.27 to 30 cP, 20 to 50 cP, 1to 30 cP, 1 to 20 cP, or 1 to 15 cP, and a concentration of the firsttherapeutic or diagnostic agent from 0.0001 to 1000 mg/mL, e.g., 100 to800, 200 to 700, 200 to 600, or 300 to 700 mg/mL. In other embodiments,the pharmaceutical composition is a dry form of particles including thetherapeutic or diagnostic agent. In a related aspect, the inventionprovides a composition, e.g., a pharmaceutical composition, includingparticles including a first therapeutic or diagnostic agent. In certainembodiments, the composition further includes a medium, where thecomposition has a viscosity from 0.27 to 200 cP, e.g., 0.27 to 100 cP,0.27 to 50 cP, 0.27 to 30 cP, 20 to 50 cP, 1 to 30 cP, 1 to 20 cP, or 1to 15 cP, and a concentration of the first therapeutic or diagnosticagent from 0.0001 to 1000 mg/mL, e.g., 100 to 800, 200 to 700, 200 to600, or 300 to 700 mg/mL. In other embodiments, the composition includesparticles in dry form.

In some embodiments, the composition includes from 5 to 90% particles byvolume, e.g., 20 to 90%, 40 to 80%, 50 to 60%, or 70 to 90%.

The administering may occur by auricular, buccal, conjunctival,cutaneous, dental, electro-osmotical, endocervical, endosinusial,endotracheal, enteral, epidural, extra amniotical, extracorporeal,infiltration, interstitial, intra-abdominal, intra-amniotical,intra-arterial, intra-articular, intrabiliary, intrabronchial,intrabursal, intracardial, intracartilaginous, intracaudal,intracavernous, intracavitary, intracerebral, intracisternal,intracorneal, intracoronal, intracoronary, intracorporus cavernosum,intradermal, intradiscal, intraductal, intraduodenal, intradural,intraepidermal, intraesophageal, intragastrical, intragingival,intraileal, intralesional, intraluminal, intralymphatical,intramedullar, intrameningeal, intramuscular, intraocular, intraovarian,intrapericardial, intraperitoneal, intrapleural, intraprostatical,intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous,intratesticular, intrathecal, intrathoracic, intratubular, intratumor,intratympanic, intrauterine, intravascular, intravenous, intravenousbolus, intravenous drip, intraventricular, intravesical, intravitreal,iontophoresis, irrigation, laryngeal, nasal, nasogastrical, occlusivedressing technique, ophthalmical, oral, oropharyngeal, parenteral,percutaneous, periarticular, peridural, perineural, periodontal, rectal,inhalation, retrobulbar, soft tissue, subarachnoidial, subconjunctival,subcutaneous, sublingual, submucosal, topical, transdermal,transmucosal, transplacental, transtracheal, transtympanic, ureteral,urethral, or vaginal administration.

In some embodiments of any aspect of the invention, the viscosity ismeasured at a shear rate in the Newtonian regime. In other embodiments,the viscosity is measured at a shear rate of 100 s⁻¹ or greater, e.g.,at 1000 s⁻¹, or greater than 1000 s⁻¹.

In some embodiments, the concentration of the first therapeutic ordiagnostic agent in the first liquid is from 0.0001 to 1000 mg/mL, e.g.,1 to 1000 mg/mL, 1 to 900 mg/mL, 1 to 500 mg/mL, 1 to 250 mg/mL, 1 to100 mg/mL, 1 to 50 mg/mL, 5 to 1000 mg/mL, 100 to 900 mg/mL, 150 to 800mg/mL, or 200 to 700 mg/mL.

In another aspect, the invention provides a method of administering afirst therapeutic or diagnostic agent to a mammal. The method includesadministering an effective amount of a suspension or a dry formulationof the particles including the therapeutic or diagnostic agent to themammal, where the first therapeutic or diagnostic agent has 0.5 to 1.0activity per unit, e.g., 0.75 to 1.0 activity per unit, or 0.9 to 1.0activity per unit (e.g., about 0.99 activity per unit). In a relatedaspect, the invention provides a composition including particlesincluding a first therapeutic or diagnostic agent has 0.5 to 1.0activity per unit, e.g., 0.75 to 1.0 activity per unit, or 0.9 to 1.0activity per unit. The composition may be a suspension of the particlesin a non-aqueous or aqueous liquid. Alternatively, the composition is indry form, e.g., a powder, such as for inhalation or needlelessinjection. The composition may be in the form of a pharmaceuticalcomposition in which the first therapeutic or diagnostic agent ispresent in an effective amount.

In some embodiments, the suspension has viscosity from 0.27 to 200 cP,e.g., 0.27 to 100 cP, 0.27 to 50 cP, 0.27 to 30 cP, 20 to 50 cP, 1 to 30cP, 1 to 20 cP, or 1 to 15 cP.

In some embodiments, the suspension includes from 5 to 90% particles byvolume, e.g., e.g., 20 to 90%, 40 to 80%, 50 to 60%, or 70 to 90%.

In some embodiments, the suspension has a concentration of the firsttherapeutic or diagnostic agent from 0.0001 to 1000 mg/mL, e.g., 100 to800, 200 to 700, 200 to 600, or 300 to 700 mg/mL.

In some embodiments, the liquid in a suspension or a dry formulationincludes a second therapeutic or diagnostic agent, e.g., at aconcentration from 0.0001 to 1000 mg/mL. The first and secondtherapeutic or diagnostic agents can be the same or different.

Definitions

The term “activity” refers to the ratio of a functional or structuralaspect of a therapeutic or diagnostic agent at two points in time. Thedenominator of the ratio corresponds to a measure of the functional orstructural aspect of the therapeutic or diagnostic agent in the feedsolution, immediately in advance of electrospray particle formation. Thenumerator of the ratio corresponds to the same measure of a functionalor structural aspect of the therapeutic or diagnostic agent at a laterpoint in time, e.g., immediately after electrospray particle formation.In certain embodiments, the activity of a protein is assessed throughSEC-HPLC or the proclivity of the protein for binding select targets.

The term “conventional electrospray” is used to refer to an electrosprayproduced with an assembly comprising a capillary tube, a hydraulic pump,a power supply, and a counter-electrode. The capillary tube andcounter-electrode are coordinated at a distance from one another anddisposed in a dielectric medium. The power supply connects the tube tothe counter-electrode in an electrical fashion, such that a relativeelectrical bias can be enforced. This produces an electric field, theagency of which facilitates an electrospray when the pump is used toeject a liquid from the end of the capillary tube.

A “dry” particle component, i.e., a dry core or a dry shell, includingthe therapeutic or diagnostic agents, has undergone a desiccation stepor series of desiccation steps, such that its moisture or solventcontent is substantially reduced in relation to that which prevailedbefore any desiccation. In some embodiments, the residual moisture orsolvent content of the dry component is less than about 10% by weight,e.g., less than about 5% by weight. Exemplary methods for themeasurement of moisture content include chemical titration methods,e.g., Karl Fischer titration involving a vacuum oven. A variety ofsolvents, including water, may also be measured using weight lossmethods involving thermal excitation. Exemplary methods includethermogravimetric analysis with Infrared spectroscopy (TGA-IR).

The term “electrospray” refers to a process by which droplets o a firstliquid are formed in a dielectric medium in the presence of an electricfield. Suitable dielectric media include vacua, air, and a second liquidwhich is at least partially immiscible with the first liquid. Thedroplets need not be electrically charged (e.g., droplets can be formedfrom electrically insulating liquids, such as oils), and the electricfield need not be the primary driving force behind the formation of thedroplets. In some embodiments, the droplets are produced by aconventional electrospray (M. Cloupeau and B. Prunet-Foch, J. AerosolSci., vol. 25, no. 6, pp. 1021-1036, 1994; J. F. de la Mora, Annu. Rev.Fluid Mech., vol. 39: 217-243, 2007). In other embodiments, the dropletsare formed by an atomizer, e.g., a pneumatic nozzle atomizer (Z. Takats,J M Wiseman, B. Gologan, and R G Cooks, Anal. Chem., 2004, 76 (14), pp.4050-4058), under an electric field. In other embodiments, the dropletsare formed by a microfluidic device, e.g., a T-junction (H. Kim, D. Luo,D. Link, D. Weitz, M. Marquez, and Z. Cheng, Appl. Phys. Lett., 91,133106, 2007), under an electric field. In some embodiments,electrospraying is a gentle method of atomization that is used togenerate the particles. In some embodiments, in electrospraying, liquidor gel (or pre-gel) is pumped from a supply through a capillary nozzleinto an electric field formed at the opening of the nozzle causing theliquid or gel (or pre-gel) to be dispersed away from the nozzle as auniform particle aerosol.

The term “electrospray particle formation” refers to the process ofelectrospraying a first liquid including a solute to form droplets andthen removing the first liquid to form particles including the solute.Removal of the first liquid, or desiccation, can be performed with oneof several methods known in the art, or some combination thereof. Insome embodiments, desiccation is carried out by contacting the dropletswith a stream of hot gas, such as in spray drying (B. Gikanga, R. Turok,A. Hui, M. Bowen, O. B. Stauch, Y. Maa, J. Pharm Sci. Tech., 2015, 69,59-73). In other embodiments, desiccation is carried out by freezing thedroplets and then subliming the first liquid, such as in spray freezedrying (S. Nanning, R. Suverkrup, A. Lamprecht, Int. J. Pharm., 2015,488, 136-153). In other embodiments, desiccation is carried out bycontacting the first liquid with a second liquid, in which it is atleast partially miscible, thereby causing the solute to precipitate,such as in certain microfluidic systems (Aniket, D. A. Gaul, D. L.Rickard, D. Needham J. Pharm. Sci., 2014, 103, 810-820).

The term “encapsulant” refers to a substance that can be dried or gelledaround a particle core to form a shell.

The term “excipient” refers to an additive to a preparation offormulation, the agency of which may be useful in achieving a desiredmodification to the characteristics of the preparation or formulation.Such modifications include, but are not limited to, physical stability,chemical stability, and therapeutic efficacy. Exemplary excipientsinclude, but are not limited to, a carbohydrate, a pH adjusting agent, asalt, a chelator, a mineral, a polymer, a surfactant, a proteinstabilizer, an emulsifier, an antiseptic, an amino acid, an antioxidant,a protein, an organic solvent, or nutrient media.

The term “feed solution” refers to a preparation of the therapeutic ordiagnostic agents in the first liquid, either as a solution, a slurry,or some other liquid form. In some embodiments, the preparation containsexcipients and, optionally, a buffer.

The term “injectability” refers to the relative ease with which a liquidformulation can be administered to a subject through the use of aninjection device. In some embodiments, the injectability is determinedby measuring the viscosity of the formulation at various shear rates. Insome embodiments, the injectability is determined by measuring thebreakaway and/or glide forces required to actuate a standard injectiondevice consisting of a syringe barrel, a plunger, and, optionally, aneedle. In some embodiments, the injectability of the suspensionformulation is superior to that of an aqueous formulation with about thesame concentration of therapeutic or diagnostic agents.

The term “injection breakaway force” refers to the force required toovercome friction between the syringe barrel and plunger of a standardinjection device before ejection of the contents of the syringe can takeplace at a steady rate. The force is applied at the outward-facing endof the syringe plunger shaft and directed along the axis of the syringebarrel. The contents of the syringe are optionally ejected through asyringe needle of prescribed gauge and length. In some embodiments, tileinjection breakaway force is measured through a load cell placed at theoutward-facing end of the syringe plunger during actuation.

The term “injection glide force” refers to the force required tomaintain a steady ejection of the contents of a standard injectiondevice. The force is applied at the outward-facing end of the syringeplunger shaft and directed along the axis of the syringe barrel. Thecontents of the syringe are optionally ejected through the tip of asyringe needle of prescribed gauge and length. In some embodiments, theinjection glide force is measured through a load cell placed at theoutward-facing end of the syringe plunger during actuation,

The term “medium” refers to a liquid in which particles are dispersed.

The term “Newtonian regime” means a range of shear rates over which thehighest and lowest values of viscosity differ by at most 1% of thehighest value.

The term “particle” refers to a quantity of therapeutic or diagnosticmaterial which, in one aspect, is in a state of matter that issubstantially solid as compared to a liquid droplet, or in a gel form.In some embodiments, the particle includes a core and a shell, where theshell is viewed as an encapsulant. In other embodiments, the particledoes not include a shell, in which case, the particle is made upentirely of a core.

The term “pharmaceutical composition” denotes a composition in which atherapeutic or diagnostic agent retains, or partially retains, itsintended biological activity or functional form, and in which onlypharmaceutically acceptable components are included.

A “pharmaceutically acceptable” component, e.g., an excipient,is acomponent which is suitable for administration to a subject, e.g., ahuman.

The term “powder formulation” refers to a solid formulation includingsolid particles in the absence of a carrier liquid. In some embodiments,the powder formulation is suitable for powder injection, e.g., with aPortal PRIME device.

The term “Rayleigh limit” refers to the specific charge, e.g., in unitsof Coulombs per kilogram, corresponding to the point at which Coulombicrepulsion overcomes the binding forces of surface tension in a drop,leading to Coulomb fission.

The term “stabilizer” refers to an excipient or a mixture of excipientswhich stabilizes the physical and/or chemical properties of thepharmaceutical formulation. In some embodiments, stabilizers prevent,e.g., degradation of the therapeutic or diagnostic agents duringelectrospray, desiccation, and/or storage of the particulate matter.Exemplary stabilizers include, but are not limited to, sugars, salts,hydrophobic salts, detergents, reducing agents, cyclodextrins, polyols,carboxylic acids, and amino acids.

A “stable” formulation refers to a formulation in which the therapeuticor diagnostic agent retains an acceptable portion of its essentialphysical and/or chemical and/or biological properties over an acceptableperiod of time. In the case of proteins and peptides, e.g., exemplarymethods of assessing stability are reviewed in (i) Peptide and ProteinDrug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York,N.Y., 1991, and (ii) Jones, A., Adv. Drug Delivery Rev, 10: 29-90(1993). In certain embodiments, chemical stability of a protein isassessed by measuring the size distribution of the sample at severalstages, These include, e.g., before particle formation (assessment ofthe feed solution), immediately after particle formation, and againafter a period of storage, where storage takes place either within or inthe absence of a suspension formulation carrier medium. In certainembodiments, the size distribution is assessed by size exclusionchromatography (SEC-HPLC).

A “sterile” formulation is aseptic or free from living microorganismsand their spores.

The term “suspension formulation” refers a liquid formulation includingsolid particles disposed within a carrier liquid in which they are notsoluble on an appropriate timescale. The particles may settle over time,i.e., the physical stability of the suspension is not indefinite, butmay be re-suspended using a form of agitation or excitation.

A “therapeutic amount” refers to an amount of a therapeutic ordiagnostic agent required to produce the desired effect.

As used herein, the terms “treat,” “treated,” and “treating” mean boththerapeutic treatment and prophylactic or preventative measures whereinthe object is to prevent or slow down (lessen) an undesiredphysiological condition, disorder, or disease, or obtain beneficial ordesired clinical results. Beneficial or desired clinical resultsinclude, but are not limited to, alleviation of symptoms; diminishmentof the extent of a condition, disorder, or disease; stabilized (i.e.,not worsening) state of condition, disorder, or disease; delay in onsetor slowing of condition, disorder, or disease progression; ameliorationof the condition, disorder, or disease state or remission (whetherpartial or total), whether detectable or undetectable; an ameliorationof at least one measurable physical parameter, not necessarilydiscernible by the patient; or enhancement or improvement of condition,disorder, or disease. Treatment includes eliciting a clinicallysignificant response without excessive levels of side effects. Treatmentalso includes prolonging survival as compared to expected survival ifnot receiving treatment.

The term “viscosity” is used to describe the property of a fluid actingto resist shearing flow. For the purposes of the present invention,viscosity can be determined using a rheometer, e.g., AR-G2 Rheometer (TAInstruments, USA), fitted with a cone and plate (2°/40 mm) at 25° C. ata specified shear rate. In certain embodiments, the viscosity ismeasured at a shear rate in the Newtonian regime. In other embodiments,the viscosity is measured at a shear rate of 100 s⁻¹ or greater, e.g.,at 1000 s⁻¹ or greater than 1000 s⁻¹.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a basic electrospray assembly. One end of a tube 3 isdisposed a distance from an electrode 4 while the other end attaches toa syringe 1, controlled by a syringe pump 2. A power supply 5 chargesthe tube 3 relative to the electrode 4, creating an electric field 6 inthe region between the two.

FIG. 2 shows a basic electrospray assembly in which a bath 7 containinga liquid 8 is disposed between an end of the tube 3 and an electrode 4.The end of the tube 3 is not immersed in the liquid 8.

FIG. 3 shows a basic electrospray assembly in which a bath 7 containinga liquid 8 is disposed between an end of the tube 3 and an electrode 4.The end of the tube 3 is immersed in the liquid 8.

FIG. 4 shows a coaxial electrospray assembly. A tube 3 connects to asyringe 1, controlled by a syringe pump 2. A second tube 11 connects toa second syringe 9, controlled by a syringe pump 10. The distal ends oftubes 3 and 11 connect to an adapter 12 that outputs a coaxial tube 13,an end of which is disposed a distance from an electrode 4. A powersupply 5 charges the coaxial tube 13 relative to the electrode 4,creating an electric field 6 in the region between the two.

FIG. 5 shows a coaxial electrospray assembly in which a bath 7containing a liquid 8 is disposed between an end of the tube 13 and anelectrode 4. The end of the tube 13 is not immersed in the liquid 8.

FIG. 6 shows a coaxial electrospray assembly in which a bath 7containing a liquid 8 is disposed between an end of the tube 13 and anelectrode 4. The end of the tube 13 is immersed in the liquid 8.

FIG. 7 shows the menisci at the end of a tube when the electric field 6is below a threshold value for electrospray. In a conventionalsingle-liquid tube 3, the meniscus of a liquid 16 does not form anelectrospray jet. In a coaxial assembly including an outer tube 14 andinner tube 15, and an outer liquid 17 and an inner liquid 16, themeniscus at the end of the assembly does not form a coaxial electrosprayjet.

FIG. 8 shows droplet formation from the end of a tube when the electricfield 6 is above a threshold value for electrospray. In a conventionalsingle-liquid tube 3, a jet 18 breaks into a droplet ensemble 20 inwhich the droplets 22 include a single liquid 16. This accompanies aflow 25 of liquid 16 through the tube 3. In a coaxial electrosprayassembly, a coaxial jet 19 breaks into an ensemble 21 of core-shelldroplets having a core 23 of liquid 16 and a shell 24 of liquid 17. Thisaccompanies a flow 26 of liquid 16 through the inner tube 15 and a flow27 of liquid 17 through the outer tube 14.

FIG. 9 shows a comparison of a solution and a suspension formulationproduced by the disclosed methods. A solution of therapeutic ordiagnostic agents 29 may be too viscous to administer with a standardinjector 28 on account of onerous intermolecular interactions 30. Incontrast, a suspension formulation 31 of particles 32 includingtherapeutic or diagnostic agents 33 may have a much lower effectiveviscosity, permitting administration with a standard injector 28.

FIG. 10 is a series of images of particles of human IgG protein thatwere formed by electrospray particle production from an aqueoussolution.

FIG. 11 is an image depicting particles of bovine serum albumin producedby electrospray particle production with an ImageJ analysis overlay. Thescale bar is 50 μm.

FIG. 12 is a series of images depicting particles of human IgG producedby electrospray particle production. Image 37 is an optical image at amagnification of 20×. Images 38 and 39 are SEM images at magnificationsof 2000× and 5000×, respectively.

FIG. 13 is a series of optical images of particles of monoclonalantibodies produced by electrospray particle production. The scale baris 50 μm.

FIG. 14 is a graph showing ELISA assay control experiment results forhuman IgG.

FIG. 15 is a graph showing a comparison of the ELISA signals forunprocessed (pre-electrospray particle formation) and electrosprayedhuman IgG.

FIG. 16 is a series of graphs showing cellular binding data for aelectrosprayed monoclonal antibody (mAb1).

FIG. 17 is a series of graphs showing cellular binding data for aelectrosprayed monoclonal antibody (mAb2).

FIG. 18 is a series of graphs showing cellular binding data for aelectrosprayed monoclonal antibody (mAb1) before and after acceleratedstorage. Particles were stored with and without a suspension medium(carrier).

FIG. 19 is a graph showing viscosity data for various aqueous andsuspension formulations at different concentrations.

FIG. 20 is an image showing human IgG particles produced by an atomizerwith the assistance of an electric field. The scale bar is 10 μm.

FIG. 21 is an image showing human IgG particles produced by an atomizerwithout the assistance of an electric field. The scale bar is 10 μm.

FIG. 22 is an image showing a family of human IgG particles including asalt which is salient at the surface of the particles. The scale bar is100 μm.

FIG. 23 is an image showing a family of human IgG particles including asalt which is salient at the surface of the particles. The scale bar is30 μm.

FIG. 24 is an image showing a family of human IgG particles including asugar which is salient at the surface of the particles. The scale bar is30 μm.

FIG. 25 is an image showing a family of human IgG particles including asugar which is salient at the surface of the particles. The scale bar is20 μm.

FIG. 26 is an image showing a family of monoclonal antibody (mAb3)particles including a sugar which is salient at the surface of theparticles. The scale bar is 100 μm.

FIG. 27 is an image showing a family of monoclonal antibody (mAb3)particles including a sugar which is salient at the surface of theparticles. The scale bar is 30 μm.

DETAILED DESCRIPTION

The present disclosure provides methods for the preparation ofelectrosprayed particles including one or more therapeutic or diagnosticagents. Particles have in the past been prepared by milling,conventional spray drying (G. Lee, Spray-Drying of Proteins. In: J. F.Carpenter and M. C. Manning (eds) Rational Design of Stable ProteinFormulations, vol 13. Springer, Boston, Mass.), freeze drying, andconventional emulsion techniques (D. Saglam, P. Venema, R. de Vries, L.M. C. Sagis, E. van der Linde, Food Hydrocolloids, 2011, 25, 1139-1148).However, each of these techniques suffers drawbacks in preparation ofparticles from certain therapeutic or diagnostic agents. Milling ofproteins, e.g., requires a first lyophilization step and subsequentgrinding which can affect bioactivity. Control over particle morphologyis also limited. Spray drying and emulsion techniques may affect proteinbiofunction due to high shear rates while the latter also presupposeshigh operating temperatures that can be harmful. A more gentle process,e.g., one that maintains bioactivity, and one whereby particlemorphology can be accurately controlled, e.g., resulting in highlymonodisperse particles, is that of electrospray.

In some embodiments, the particles do not have a shell. In someembodiments, the particles have a liquid, solid, or gel core and ashell. The liquid, solid, or gel includes the one or more therapeutic ordiagnostic agents. In some embodiments, the therapeutic or diagnosticagents can be in the shell. The therapeutic or diagnostic agent may bedissolved or suspended in the liquid or gel, or otherwise carried by theliquid or gel, e.g., in a slurry. The electrosprayed particles can beincorporated into a medium to form a pharmaceutical composition. In someembodiments, the pharmaceutical compositions are high concentrationcolloidal suspensions or slurries having low viscosity, e.g., <50 cP orhigher (Dias, C. et al. AAPS PharmSciTech. 2015, 16, 1107), whilemaintaining the stability and controlled drug release rates of thetherapeutic or diagnostic agents. In some embodiments, the presentinvention allows for higher doses of therapeutic or diagnostic agents tobe delivered while minimizing the delivery volume, shorteningadministration time, and/or reducing pain. In other embodiments, itprovides for a powder composition of therapeutic or diagnostic agentsthat can be stored for periods of time in a stable fashion.

The particles can be formed by electrospraying a first liquid includinga therapeutic or diagnostic agent to form droplets and removing (e.g.,evaporating) the first liquid to produce particles from the droplets.The particles are solid in at least one aspect, e.g., they may have asolid shell and a liquid core. The particles can be suspended in anon-aqueous or aqueous liquid, thereby forming a non-aqueous or aqueoussuspension. Alternatively, the particles can be employed in a dry form,e.g., as a powder. Electrospray permits high throughput gentlepreparation and compatibility with highly viscous feed solutions.Importantly, the process of generating non-aqueous or aqueoussuspensions with therapeutic or diagnostic agents does not significantlyalter the structure or bioactivity of the agents. In addition, in someembodiments, the present invention allows for the delivery of higherdoses of therapeutic or diagnostic agents while minimizing the deliveryvolume, shortening administration time, and/or reducing pain.

Suspension Concept

A pharmaceutical suspension formulation is formed in some embodiments toimprove the injectability of certain therapeutic or diagnostic agents.Specifically, the suspension may exhibit lower viscosity than an aqueoussolution of comparable therapeutic or diagnostic agent loading, therebyreducing the forces required to administer the suspension with astandard injector device, i.e., the breakaway and glide forces.Conceptually, particles in suspension provide a means for replacing theintermolecular interactions that prevail in regular solution, e.g.,aqueous solution, with less onerous effects, e.g., excluded volumeeffects. In some embodiments, this permits the performance of thesuspension to approximately obey the Einstein Equation for the viscosityof solutions (E. W. J. Mardles, Nature, 1940, 145, 970):

η=η₀(1+2.5ϕ)

where η is the apparent viscosity of the suspension, n₀ is the viscosityof the suspension carrier medium, and ϕ is the volume fraction of thesolutes or particles. To aid with the conceptual understanding, FIG. 9presents a comparison of a conventional approach to protein deliveryinvolving a high viscosity solution 29 (top drawing). The electrosprayparticle suspension technique 31 of the current invention is shown withparticles 32 including a protein 33 (bottom drawing). Although thistechnique of the invention involves regions in which the local viscositymay be extremely high, i.e., within the particles, where the viscositymay be exceedingly large if the particle is a dry solid or concentratedliquid, the average macro-scale viscosity of the injectable suspensionformulation is reasonably low, such that it may be administered with astandard injection device 28. Note that the continuous phase of theinjectable suspension formulation, the carrier medium, may contain anon-zero concentration of a therapeutic or diagnostic agent. Thistherapeutic or diagnostic agent may or may not be the same therapeuticor diagnostic agent which is included the particles.

In other embodiments, particularly those involving high volume fractionϕ, the performance of the suspensions approximately obeys otherequations such as the Krieger-Dougherty equation or the Frankel-Acrivosequation (S. ueller, E. W. Llewellin, H. M. Mader, Proc. Royal Soc. A,2010, 466, 2116), amongst others.

In certain embodiments, the suspension formulation provides a means ofenhancing the stability of certain therapeutic or diagnostic agents at agiven concentration, e.g., as compared to an aqueous formulation, andimproving the injectability concurrently. In other embodiments, in whichthe injectability is not necessarily improved, the suspensionformulation enhances the stability properties of the therapeutic ordiagnostic at a given concentration. In still other embodiments, powderformulations enhance the stability of certain therapeutic or diagnosticagents, e.g., as compared to an aqueous formulation.

Particle

The particles can have diameters from 0.1 to 1000 μm. For example, arange of 0.1 to 90 μm may be delivered by a 25 gauge needle. Largerparticles, e.g., within the range from 90 to 230 μm, may be of use inconnection with smaller gauge needles or other delivery routes ormodalities. A lower range of 0.1 to 1 μm is of interest in certainembodiments. The particles can have a dispersity index from 0.05 to 0.9.Methods of measuring the particle size and distribution include imagingflow cytometry and image analysis of scanning electron micrographs ofthe particles in which an average spherical radius or diameter iscalculated on the basis of the cross-sectional areas of the particlesprojected onto the plane of the image.

The particles may include both a core and a shell. In some embodiments,the particles include a core but not a shell. The core is a gel core ordry solid-state core when no shell is present but may exist in theliquid state when the particles include a gel shell or dry solid-stateshell. The morphology of the particles is approximately spherical,mushroom-like, or raisin-like, among potentially other morphologies(FIG. 10), depending on the properties of the electrospray feed solutionand the desiccation conditions. In some embodiments the particlesurfaces may have wrinkles or crenellations.

In some embodiments, the particles exhibit a skeletal density from about1 to 6 g/cm³, e.g., from about 1 to 5 g/cm³, from about 1 to 3 g/cm³,from about 1 to 2 g/cm³, from about 1 to 1.5 g/cm³, or from about 1.1 to1.4 g/cm³. Exemplary methods of density measurements include gasdisplacement pycniometry.

In some embodiments, residual quantities of the first liquid in theparticles after desiccation are from 0 to 10% by weight, e.g., from 0 to5% by weight, from 0 to 3% by weight, or from 0 to 1% by weight.Exemplary methods of measuring residual solvent content include KarlFischer titration and various weight-loss methods.

In some embodiments, the particles may exhibit a porosity from about 0to 50%, e.g., from about 0 to 10%, from about 0 to 5%, from about 0 to1%, from about 0 to 0.5%, from about 0 to 0.1%, or from about 0 to0.01%. Exemplary pore size measurements include scanning electronmicroscopy (SEM), transmission electron microscopy (TEM), and confocallaser scanning microscopy analysis. The specific surface area of porousmicro- and nanospheres may also be investigated by nitrogenadsorption/desorption analysis and a Branauer-Emmett-Teller adsorptionmodel. In embodiments where the pore sizes are sufficiently large,mercury-intrusion porosimetry may be employed.

In some embodiments, the particles have a residual net electrical chargeof either polarity, i.e., net positive or net negative charge. In termsof magnitude, the particles may have from 0 to 10 billion charges, e.g.,from 0 to 100 million charges, from 0 to 1 million charges, from 0 to0.01 million charges, or from 0 to 100 charges. The magnitude of acharge is defined as the magnitude of charge carried by an electron,i.e., the elementary charge, 1.6×10⁻¹⁹ Coulombs. Exemplary methods ofmeasuring particle charge include those involving the analysis ofparticle motion in response to an externally applied electric field. Insome cases, this is done while particles are suspended in an insulatingliquid such as oil.

In certain embodiments, the therapeutic or diagnostic agents have a zetapotential from about −90 to 90 mV; e.g., from about −60 to 60 mV, fromabout −40 to 40 mV, from about −20 to 20 mV, or from about −5 to 5 mV.Exemplary methods of measuring zeta potential include reconstituting thetherapeutic or diagnostic agents by dissolving the particles in waterand analyzing the solution by electrophoretic light scattering. This issimilar to a dynamic light scattering (DLS) measurement which isperformed in the presence of a positive or negative electric field.

In some embodiments, sub-visible particles (SVPs) which persist uponreconstitution of the particles are present in quantities from about 0to 10,000 per mL, e.g., from 0 to 6,000 per mL, from 0 to 1,000 per mL,from 0 to 500 per mL, from 0 to 250 per mL, from 0 to 100 per mL, orfrom 0 to 10 per mL. Exemplary methods of measuring SVPs includemicro-flow imaging in which the therapeutic or diagnostic agent isreconstituted and diluted to a concentration of about 1 mg/mL.

In some embodiments, the particles include a loading of therapeutic ordiagnostic agents from 1 to 100 wt %, e.g., from 50 to 100 wt %, from 75to 100 wt %, from 90 to 100 wt %, from 95 to 100 wt %, from 99 to 100 wt%, or from 99.9 to 100 wt %. At these loadings the therapeutic ordiagnostic agents retain from 0.5 to 1.0 activity during electrosprayparticle formation, e.g., from 0.75 to 1.0 activity, from 0.9 to 1.0activity, from 0.95 to 1.0 activity, from 0.99 to 1.0 activity, or from0.999 to 1.0 activity. This includes the activity retained throughprimary desiccation and, in some cases, secondary desiccation.

In some embodiments, the dissolution or reconstitution of the particlesprovides less than 10% of aggregates of the diagnostic or therapeuticagent, e.g., a protein, (e.g., less than 8%, less than 5%, less than 4%,less than 3%, or less than 1%) as measured, e.g., by HPLC.

In some embodiments, the particles are flowable. The Hausner ratio maybe from 1.0 to greater than 3.0, e.g., from 1.0 to 3.0, from 1.0 to 2.0,from 1.0 to 1.70 (e.g., very poor), from 1.0 to 1.59, from 1.0 to 1.35,from 1.0 to 1.25, or from 1.0 to 1.11 (e.g., excellent). Exemplarymethods of measuring the flowability of a powder include the tappeddensity method (Carr R L. Chem. Eng., 1965; 72:163-168). Bulk densitymay first be obtained by adding a known mass of powder to a graduatedcylinder. The density can be calculated as mass/volume. The same samplemay then be mechanically tapped until further volume change is notobserved. The tapped density can then be calculated as mass divided bythe final volume of the powder. A comparison of tapped and bulk densitymay be used to index the ability of the powder to flow. In particular,the Hausner ratio (unsettled apparent volume or bulk volume, V₀, dividedby the final tapped volume, V_(f)) is a measure of the product's abilityto settle and permits an assessment of the relative importance ofinterparticulate interactions. These interactions are less significantin free flowing powders. The bulk and tapped densities for such freeflowing powders are close in value, such that the Hausner ratio is closeto 1.0.

In some embodiments, the particles have one or more of the followingcharacteristics: a size from 1 to 50 μm; a solid core; a gel or solidshell; a density from 1 to 1.5 g/cm³; a residual solvent content from 0to 3 wt %; a porosity from 0 to 10%; a net electrical charge of eitherpolarity, i.e., positive or negative charge, from 0 to 1 millioncharges; therapeutic or diagnostic components with a zeta potential from−60 to 60 mV; SVPs from 0 to 1,000 per mL upon reconstitution; atherapeutic or diagnostic agent loading from 50 to 100 wt % in which theactivity of the therapeutic or diagnostic agents is from 0.9 to 1.0 uponreconstitution; less than 10% aggregates upon reconstitution; and/or aHausner ratio between 1.0 and 1.35, or between 1.0 and 1.11.

Particles can be stored and formulated for delivery in various devices.In some embodiments, the device is a subcutaneous administration device,such as a pre-filled syringe. In some embodiments, the inventionprovides a method of making an article of manufacture including fillinga container with a suspension formulation. The container in the articleof manufacture may include syringes (e.g., pre-filled syringes),autoinjectors, bottles, vials (e.g., dual chamber vials), and testtubes. The container may hold the suspension formulation, and the labelon, or associated with, the container may indicate directions for use.The article of manufacture may further include other materials desirablefrom a commercial and user standpoint, including, e.g., other buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use.

Particle Core

The core of each particle typically includes one or more therapeutic ordiagnostic agents. The core is a solid-state dry core when no shell ispresent but may exist in the liquid state when the particle includes agel shell or solid-state dry shell. When a shell is present, the shellmay include the therapeutic or diagnostic agent, while the core doesnot.

The therapeutic or diagnostic agent or agents may be dissolved orsuspended in the electrospray feed solution, a first liquid, prior toparticle formation. The concentration of the therapeutic or diagnosticagent in the first liquid can be in the range of 0.0001 to 1000 mg/mL.The liquid can be aqueous or an organic solvent, a hydrogel, an ionogel,or a combination thereof. The liquid is, for example, water, 0.9%saline, lactated Ringer's solution, dextrose 5% or a buffer. In someembodiments, the buffer is acetate buffer, histidine buffer, succinatebuffer, HEPES buffer, tris buffer, carbonate buffer, citrate buffer,phosphate buffer, glycine buffer, barbital buffer, and cacodylatebuffer. Organic solvents, hydrogels, and ionogels are described herein.The liquid can further include, e.g., a carbohydrate, a pH adjustingagent, a salt, a chelator, a mineral, a polymer, a surfactant, a proteinstabilizer, an emulsifier, an antiseptic, an amino acid, an antioxidant,a protein, an organic solvent, and/or nutrient media. In someembodiments, each of the other components is, independently, at 0.0001to 99% (w/v) of sprayed liquid, e.g., at 0.0001 to 90% (w/v), at 0.0001to 50% (w/v), at 0.0001 to 10% (w/v), at 0.0001 to 1% (w/v), or at0.0001 to 0.1% (w/v). One of ordinary skill in the art would be able todetermine an appropriate amount of the other components in the sprayedliquid. In some embodiments, the carbohydrate is dextran, trehalose,sucrose, agarose, mannitol, lactose, sorbitol, or maltose. In someembodiments, the pH adjusting agent is acetate, citrate, glutamate,glycinate, histidine, lactate, maleate, phosphate, succinate, tartrate,bicarbonate, aluminum hydroxide, phosphoric acid, hydrochloric acid,DL-lactic/glycolic acids, phosphorylethanolamine, tromethamine,imidazole, glyclyglycine, or monosodium glutamate. In some embodiments,the salt is sodium chloride, calcium chloride, potassium chloride,sodium hydroxide, stannous chloride, magnesium sulfate, sodiumglucoheptonate, sodium pertechnetate, or guanidine hydrochloride. Insome embodiments, the chelator is disodium edetate. In some embodiments,the mineral is calcium, zinc, or titanium dioxide. In some embodiments,the polymer is propyleneglycol, glucose star polymer, silicone polymer,polydimethylsiloxane, polyethylene glycol, carboxymethylcellulose,poly(glycolic acid), poly(lactic-co-glycolic acid), or polylactic acid.In some embodiments, the surfactant is polysorbate, magnesium stearate,sodium dodecyl sulfate, polyethylene glycol nonylphenyl ether (Triton™N-101), glycerin, or polyoxyethylated castor oil. In some embodiments,the protein stabilizer is acetyltryptophanate, caprylate, orN-acetyltryptophan. In some embodiments, the emulsifier is selected frompolysorbate 80, polysorbate 20, sorbitan monooleate, ethanolamine,polyoxyl 35 castor oil, poloxyl 40 hydrogenated castor oil, carbomer1342, a corn oil-mono-di-triglyceride, a polyoxyethylated oleicglyceride, or a poloxamer. In some embodiments, the antiseptic isphenol, m-cresol, benzyl alcohol, 2-phenyloxyethanol, chlorobutanol,neomycin, benzethonium chloride, gluteraldehyde, or beta-propiolactone.In some embodiments, the amino acid is alanine, aspartic acid, cysteine,isoleucine, glutamic acid, leucine, methionine, phenylalanine,pyrrolysine, serine, selenocysteine, threonine, tryptophan, tyrosine,valine, asparagine, L-arginine, histidine, glycine, or glutamine, e.g.,asparagine, L-arginine, histidine, glycine, or glutamine. In someembodiments, the antioxidant is glutathione, ascorbic acid, cysteine, ortocopherol. In some embodiments, the protein is protamine, protaminesulfate, or gelatin. In some embodiments, the organic solvent may bedimethyl sulfoxide or N-methyl-2-pyrrolidone. In some embodiments, thepreservative is methyl hydroxybenzoate, thimerosal, parabens,formaldehyde, or castor oil. In some embodiments, the liquid can furtherinclude adenine, tri-n-butyl phosphate, octa-fluoropropane, whitepetrolatum, or p-aminophenyl-p-anisate. In some embodiments, the organicsolvent may be dichloromethane, dimethyl sulfoxide, urea, sarcosine,methanol, formic acid, acetic acid, ethyl acetate, acetonitrile,acetone, methyl acetate, diethyl ether, hydrazine, ethyl nitrate,butanol, dimethoxyethane, methyl tert-butyl ether, triethylamine, or anycombination thereof.

Particle Shell

Generally, any excipient is suitable as a shell material. Exemplaryexcipients include, but are not limited to, sugars, salts, and aminoacids. Therapeutic agents, diagnostic agents, and biocompatible polymersmay also be used to form the shell. This includes small molecule drugs.Non-limiting examples of hydrophilic biocompatible polymers includepoly(vinyl alcohol), poly(acrylic acid), poly(acrylamide), poly(ethyleneoxide), or co-polymers or combinations of any two or more of them.Hydrophilic polymers may be modified to adjust their characteristics.The shell component may alternatively or additionally include one ormore biocompatible hydrophobic polymers. Hydrophobic polymers may bemodified to adjust their characteristics. Non-limiting examples ofhydrophobic polymers include polycaprolactam, poly(lactic acid),poly(glycolic acid), polycaprolactone, PLGA or co-polymers, orcombinations of any two or more of them. In some embodiments, a PLGA(50:50) polymer is used as a shell to encapsulate an antibody in anamount just below its solubility limit. The polymer also may be preparedas a function of PLGA at various lactic acid-glycolic acid ratios, aswell as be co-polymer with other polymers, e.g., chitosan, cellulose,etc.

The thickness of the particle shell may range from 0 to 90% of thediameter of the particle in some embodiments. The shell does not have tobe uniform of fully formed for encapsulation. In some embodiments theinterface between the shell and the core is partially blended, such thata clear line of demarcation does not exist. Moreover, one or moretherapeutic or diagnostic agents, as described herein, can be includedin the particle shell. The therapeutic or diagnostic agents can be thesame or different as those in the core. The concentration of thetherapeutic or diagnostic agent in the shell may be in the range 0.0001to 300 mg/mL.

Core-Shell Ratio

For those embodiments in which the particle includes a shell, acore-shell volume ratio between 1:99 vol% and 99:1% are expected to bemost useful, e.g., about 10:90 vol% or about 90:10 vol% or about 95:5vol%. Complete coverage is not always required for sufficientencapsulation. In certain circumstances, e.g., for highly concentratedcores, thick shells can be beneficial. The core-shell ratio may beuseful in the modulation of the release kinetics of the therapeutic ordiagnostic agent or agents. In certain embodiments, it is advantageousto have a polydisperse system, e.g., for lowering the viscosity of asuspension formulation. In this instance a variety of core-shell ratiosmay be of interest.

Electrospray Particle Formation

Electrospray is a process by which droplets of a first liquid are formedin a dielectric medium in the presence of an electric field. Exemplarydielectric media include vacuo, air, a second liquid that is a suitableelectrical insulator in which the first liquid is at least partiallyimmiscible, and combinations thereof. The first liquid does not need tobe electrically conductive, and the droplets do not need to possess netelectrical charge (e.g., droplets can be formed from electricallyinsulating liquids, such as ohs). Furthermore, the electric field actingon the first liquid need not be the primary driving force behind theformation of the droplets. In some embodiments, droplets are formedprimarily on account of electrostatic interactions between the firstliquid and the electric field, such as in conventional electrospray. Inother embodiments, the electric field acts to assist in the formation ofdroplets by a primary droplet formation device, playing an ancillaryrole and in some instances modifying the properties of the droplets.Devices include rotary atomizers, pneumatic nozzle atomizers, ultrasonicnozzle atomizers, sonic nozzles, microfluidic T-junctions, microfluidicY-junctions, microcapillaries, etc. In all embodiments, the electricfield is effectuated by enforcing a potential difference between thefirst liquid and an electrode which is disposed in the dielectricmedium.

The potential difference between the location at which droplets arefirst produced and the electrode, as measured within the dielectricmedium separating the electrospray source and the electrode, is from0.001 and 100,000 V, e.g., from 1 to 50,000 V, 100 to 25,000 V, or 1,000to 10,000 V. As a fraction of the Rayleigh limit, the droplets in thisfield may on average be charged from 0 to 1, e.g., from 0.1 to 1.0, from0.2 to 1.0, from 0.3 to 1.0, from 0.4 to 1.0, or from 0.5 to 1.0. Insome embodiments the droplets are charged to beyond the Rayleigh limitto induce Coulomb fission. The droplets can be desiccated through any ofseveral techniques known throughout the literature and then collectedfor use. Exemplary collectors include, but are not limited to, electrodeplates (N. Bock, T. R. Dargaville, M. A. Woodruff, Prog. Polym. Sci.,2012, 37, 1510-1551), liquid baths (FIGS. 2, 3, 5, 6) (U.S. Pat. No.8,939,388), electrostatic precipitators (Y. A. Haggag, A. M. Faheem,Front. Pharmacol., 2015, 6, 140), and cyclones (J. Bogelein, G. Lee,Int. J. Pharm., 2010, 401, 68-71).

The electrospray technique is perhaps most widely known for itsutilization in biological mass spectrometers (J. Fenn, M. Mann, C. KaiMeng, S. Fu Wong, and C. M. Whitehouse, Science, vol. 246, no. 4926, pp.64-71, October 1989), where its properties of soft atomization havehelped to enable the analysis of molecules with high molecule weight.The canonical electrospray apparatus includes a tube and an electrodewhich are coordinated such that one end of the tube is located at adistance from the electrode (FIG. 1). The tube carries a flow of liquidthat is driven by a displacement pump, e.g., a syringe pump, or a sourceof pressurized gas, e.g., a compressed gas bottle. A power supplycharges the liquid in the tube relative to the electrode, creating anelectric field in the region between the distal end of the tube and theelectrode. Charges accumulate in the liquid meniscus at the end of thetube in proportion to the strength of this field (FIG. 7). When thefield reaches a certain critical strength, the force of electrostatictraction acting on the charge is sufficient to overcome the surfacetension forces of the meniscus (FIG. 8). In some embodiments, thisresults in a morphologically stable reconfiguration of the meniscus thatis typified by a conewith a half-angle of about 49.2 degrees. Thisso-called Taylor cone, characteristic of conventional electrospraydroplet formation, anchors at its tip a thin jet which extends brieflytoward the electrode as it breaks into a train of uniform chargeddroplets. These droplets propagate toward the electrode through theagency of the field before they are eventually intercepted. In otherembodiments, the increase in electrostatic traction over surface tensionforces results in a pulsating meniscus that produces droplets viadripping. During each cycle of pulsation the meniscus extends towardsthe electrode in the form of a jet that eventually detaches from thebase of the meniscus. The detached jet forms either a single droplet orbreaks into an ensemble of droplets as the meniscus recoils inpreparation for a subsequent cycle. In still other embodiments,disparate modes of electrospray prevail. The exact mode through whichelectrified droplets are produced is generally dependent upon at leastthe conductivity, polarizability, viscosity, surface tension coefficientof the electrosprayed liquid, and electric field geometry (M. Cloupeauand B. Prunet-Foch, Electrostatic spraying of liquids: Main functioningmodes, J. Electrostatics, 25, 165-184, 1990; M. Cloupeau and B.Prunet-Foch, J. Aerosol Sci., vol. 25, no. 6, pp. 1021-1036, 1994).

In some embodiments, the electrospray technique is utilized bycomplementing an atomizer, a microfluidic device, or some other primarydrop formation device with an electric field to form electrospraydroplets. In some embodiments, this can reduce the amount of energy thatthe primary device contributes to the formation of each drop. In theinstance of an ultrasonic atomizer, e.g., the presence of the electricfield may in some cases reduce the minimum power required by theultrasonic generator to produce drops. This can be advantageous in thatit mitigates select effects, e.g., cavitation, which may have an impacton the integrity of certain therapeutic or diagnostic agents in thefirst liquid (S. Vonhoff, The Influence of Atomization Conditions onProtein Secondary and Tertiary Structure During Microparticle Formationby Spray-Freeze-Drying, PhD Thesis, Univ. of Erlangen-Nuremberg, 2010).In certain embodiments, the electric field may similarly reduce theaverage droplet size and/or narrow the droplet dispersity relative towhat can be achieved in its absence.

Desiccation of the droplets, i.e., removal of the first liquid toproduce dry particles, is performed through any of several methods knownthroughout the art. These include, but are not limited to, warm gasevaporation, freeze drying, critical point drying, emulsion solventevaporation, emulsion solvent diffusion (U.S. Pat. Nos. 8,013,022;8,512,754), and combinations thereof. In some embodiments, the primarydesiccation step is followed by a secondary desiccation step such aslyophilization or vacuum desiccation intended to further reduce residualquantities of the first liquid within the particles. In someembodiments, residual quantities of the first liquid in the particlesafter primary or secondary desiccation are from 0 and 10% by weight,e.g., from 0 to 5% by weight, or from 0 to 3% by weight, or from 0 to 1%by weight.

In some embodiments, the agency of the electric field is such that areversible or irreversible charge is induced on the therapeutic ordiagnostic agents in a droplet of the first liquid, i.e., the agents areionized (Anal. Chem., 2005, 77, 5370). This effect may be modulated bycontrolling certain electrospray parameters such as the size of thedrops and their composition. In some embodiments, the inclusion ofcertain excipients, e.g., amino acids, stabilizes the chargedtherapeutic or diagnostic agents. In other embodiments, the excipientcarries the charge preferentially, such that the therapeutic ordiagnostic agents are less susceptible to ionization. In someembodiments, charge on the drops is reduced or even completelyneutralized during desiccation and/or contact between the particle andan electrode. In other embodiments, portions of this charge areintentionally preserved by preventing direct contact between theparticle and an electrode.

In some embodiments, the agency of the electric field is such that freecharges and/or polar molecules move to the surface of the droplet of thefirst liquid preferentially on account of Coulombic effects. The formerphenomenon, the localization of free charges at the interface betweenthe first liquid and the dielectric medium in which the droplets areformed, produces a layer of surface charge. In some embodiments, sucheffects are leveraged to influence the structure and/or surfaceproperties of the droplet and/or particle. In some embodiments, e.g.,coordination of the first liquid near the surface of the dropletfacilitates faster removal of the first liquid and in some cases atlower temperatures. The ability to reach low residual moisture contentwith primary desiccation may also be improved. This may be particularlybeneficial when desiccating with a warm gas stream, where thetemperature of the gas stream is in some cases correlated withdegradation of the therapeutic or diagnostic agents.

In some embodiments, the agency of the electric field is such that freecharges and/or polar molecules move to the surface of the droplet of thefirst liquid preferentially on account of Coulombic effects, and thetherapeutic or diagnostic agent crystallizes. Crystal nucleation of theagent may be controlled to obtain a desired polymorph preferentially (A.Ziabicki, L. Jarecki, Macromolecular Symposia, 1996, 104, 65-87).

In some embodiments, core-shell particles are produced by coaxialelectrospray (FIG. 4). In this case a second liquid including adissolved encapsulant or shell material is provided with the firstliquid during electrospraying. This is typically achieved by flowing thesecond liquid through an annular tube that is coordinated coaxially withrespect to a tube through which the first liquid flows. At end of thiscoaxial tube arrangement, an electrospray is formed in which dropletsinclude a core of the first liquid and a shell of the second liquid.Desiccation of the droplets may proceed through any of the usualpathways. In other embodiments, core-shell particles are formed from anelectrospray of only a first liquid by leveraging the proclivity ofcertain polar molecules and free charges to arrange themselves at thesurface of the droplet. In certain instances, this produces alocalization of the therapeutic or diagnostic agents, either towards thecore of the droplet or its surface, that can be preserved duringdesiccation. In some embodiments, this involves a deterministicstratification of various agents (e.g., therapeutic agents, diagnosticagents, excipients) throughout the thickness of the particle. In certainembodiments, non-therapeutic components, such as a salt (e.g., NaCl) ora sugar (e.g., sucrose) are driven to the surface, preferentially withthe electric field, to form a thin shell around the particle,crystalline or otherwise. This shell may have protective effects orprovide a measure of control over pharmacokinetics. In otherembodiments, portions of the agents may be localized at the particlesurface without necessarily forming a uniform or continuous shell (FIG.22, FIG. 23).

Therapeutics

Exemplary therapeutic or diagnostic agents are nucleic acids,oligonucleotides, antibodies, amino acids, peptides, proteins, cells,bacteria, gene therapeutics, genome engineering therapeutics, epigenomeengineering therapeutics, carbohydrates, chemical drugs, contrastagents, magnetic particles, polymer beads, metal nanoparticles, metalmicroparticles, quantum dots, antioxidants, antibiotic agents, hormones,nucleoproteins, polysaccharides, glycoproteins, lipoproteins, steroids,analgesics, local anesthetics, anti-inflammatory agents, anti-microbialagents, chemotherapeutic agents, exosomes, outer membrane vesicles,vaccines, viruses, bacteriophages, adjuvants, vitamins, minerals,organelles, and combinations thereof (Table 1). Therapeutic anddiagnostic agents may have a molecular weight of 20 to 200 kDa, e.g., 40to 150 kDa. The concentration of the therapeutic or diagnostic agent inthe droplet is typically at least 1 mg/mL, e.g., at least 5 mg/mL, atleast 10 mg/mL, at least 50 mg/mL, at least 100 mg/mL, or at least 500mg/mL. The first therapeutic or diagnostic agent in the droplets mayhave 0.5 to 1.0 activity per unit, 0.75 to 1.0 activity per unit, 0.9 to1.0 activity per unit, 0.95 to 1.0 activity per unit, or 0.99 to 1.0activity per unit. Activity is measured relative to the same therapeuticor diagnostic agent prior to being electrosprayed.

TABLE 1 Various therapeutic and diagnostic agents in the particles andtheir concentrations. Therapeutic/diagnostic agent Concentration range(mg/mL) proteins 20-1500 (e.g., 20-600) (or crystalline density, ifhigher) peptides 20-1500 (e.g., 20-600) (or crystalline density, ifhigher) chemical drugs 0.0001-2000 (e.g., 0.0001-1000) (or crystallinedensity, if higher) magnetic particles 0.001-5400 (e.g., 0.001-500)(iron oxide density) carbohydrates 0.001-400 nucleic acids 0.001-100

In some embodiments of any of the foregoing methods, the therapeutic anddiagnostic agent is an antibody. In some embodiments, the antibody is3F8, Abagovomab, Abciximab, Abituzumab, Abrilumab, Acritumomab,Actoxumab, Adalimumab, Adalimumab-atto, Adecatumumab, Ado-trastuzumabemtansine, Aducanumab, Afasevikumab, Afelimomab, Afutuzumab, Alacizumabpegol, ALD518, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab,Anatumomab mafenatox, Anetumab ravtansine, Anifrolumab, Anrukinzumab,Apolizumab, Arcitumomab, Ascrinvacumab, Aselizumab, Atezolizumab,Atinumab, Atlizumab, Atorolimumab, Avelumab, Bapineuzumab, Basiliximab,Bavituximab, Bectumomab, Begelomab, Belimumab, Benralizumab,Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab,Bimagrumab, Bimekizumab, Bivatuzumab mertansine, Bleselumab,Blinatumomab, Blontuvetmab, Blosozumab, Bococizumab, Brazikumab,Brentuximab vedotin, Briakinumab, Brodalumab, Brolucizumab,Brontictuzumab, Burosumab, Cabiralizumab, Canakinumab, Cantuzumabmertansine, Cantuzumab ravtansine, Caplacizumab, Capromab pendetide,Carlumab, Carotuximab, Catumaxomab, cBR96-doxorubicin immunoconjugate,Cedelizumab, Cergutuzumab amunaleukin, Certolizumab pegol, Cetuximab,Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab,Clivatuzumab tetraxetan, Codrituzumab, Coltuximab ravtansine,Conatumumab, Concizumab, Crenezumab, Crotedumab, CR6261, Dacetuzumab,Daclizumab, Dalotuzumab, Dapirolizumab pegol, Daratumumab, Dectrekumab,Demcizumab, Denintuzumab mafodotin, Denosumab, Depatuxizumab mafodotin,Derlotuximab biotin, Detumomab, Dinutuximab, Diridavumab, Domagrozumab,Dorlimomab aritox, Drozitumab, Duligotumab, Dupilumab, Durvalumab,Dusigitumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab,Efalizumab, Efungumab, Eldelumab, Elgemtumab, Elotuzumab, Elsilimomab,Emactuzumab, Emibetuzumab, Emicizumab, Enavatuzumab, Enfortumab vedotin,Enlimomab pegol, Enoblituzumab, Enokizumab, Enoticumab, Ensituximab,Epitumomab cituxetan, Epratuzumab, Erenumab, Erlizumab, Ertumaxomab,Etaracizumab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab,Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, Felvizumab,Fezakinumab, Fibatuzumab, Ficlatuzumab, Figitumumab, Firivumab,Flanvotumab, Fletikumab, Fontolizumab, Foralumab, Foravirumab,Fresolimumab, Fulranumab, Futuximab, Galcanezumab, Galiximab, Ganitumab,Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin, Gevokizumab,Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, Guselkumab,Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Idarucizumab, Igovomab,IMAB362, Imalumab, Imciromab, Imgatuzumab, Inclacumab, Indatuximabravtansine, Indusatumab vedotin, Inebilizumab, Infliximab,Infliximab-dyyb, Intetumumab, Inolimomab, Inotuzumab ozogamicin,Ipilimumab, Iratumumab, Isatuximab, Itolizumab, Ixekizumab, Keliximab,Labetuzumab, Lambrolizumab, Lampalizumab, Lanadelumab, Landogrozumab,Laprituximab emtansine, Lebrikizumab, Lemalesomab, Lendalizumab,Lenzilumab, Lerdelimumab, Lexatumumab, Libivirumab, Lifastuzumabvedotin, Ligelizumab, Lilotomab satetraxetan, Lintuzumab, Lirilumab,Lodelcizumab, Lokivetmab, Lorvotuzumab mertansine, Lucatumumab,Lulizumab pegol, Lumiliximab, Lumretuzumab, Mapatumumab, Margetuximab,Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab,Milatuzumab, Minretumomab, Mirvetuximab soravtansine, Mitumomab,Mogamulizumab, Monalizumab, Morolimumab, Motavizumab, Moxetumomabpasudotox, Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomabestafenatox, Naratuximab emtansine, Narnatumab, Natalizumab,Navicixizumab, Navivumab, Nebacumab, Necitumumab, Nemolizumab,Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentan,Obiltoxaximab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odulimomab,Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab,Ontuxizumab, Opicinumab, Oportuzumab monatox, Oregovomab, Orticumab,Otelixizumab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab,Pagibaximab, Palivizumab, Pamrevlumab, Panitumumab, Pankomab,Panobacumab, Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab,Patritumab, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab,Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab,Plozalizumab, Pogalizumab, Polatuzumab vedotin, Ponezumab, Prezalizumab,Priliximab, Pritoxaximab, Pritumumab, PRO 140, Quilizumab, Racotumomab,Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranibizumab,Raxibacumab, Refanezumab, Regavirumab, Reslizumab, Rilotumumab,Rinucumab, Risankizumab, Rituximab, Rivabazumab pegol, Robatumumab,Roledumab, Romosozumab, Rontalizumab, Rovalpituzumab tesirine,Rovelizumab, Ruplizumab, Sacituzumab govitecan, Samalizumab,Sapelizumab, Sarilumab, Satumomab pendetide, Secukinumab, Seribantumab,Setoxaximab, Sevirumab, Sibrotuzumab, SGN-CD19A, SGN-CD33A, Sifalimumab,Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Sofituzumab vedotin,Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab,Suvizumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab,Tamtuvetmab, Tanezumab, Taplitumomab paptox, Tarextumab, Tefibazumab,Telimomab aritox, Tenatumomab, Teneliximab, Teplizumab, Teprotumumab,Tesidolumab, Tetulomab, Tezepelumab, TGN1412, Ticilimumab,Tildrakizumab, Tigatuzumab, Timolumab, Tisotumab vedotin, TNX-650,Tocilizumab, Toralizumab, Tosatoxumab, Tositumomab, Tovetumab,Tralokinumab, Trastuzumab, Trastuzumab emtansine, Tregalizumab,Tremelimumab, Trevogrumab, Tucotuzumab celmoleukin, Tuvirumab,Ublituximab, Ulocuplumab, Urelumab, Urtoxazumab, Ustekinumab,Utomilumab, Vadastuximab talirine, Vandortuzumab vedotin, Vantictumab,Vanucizumab, Vapaliximab, Varlilumab, Vatelizumab, Vedolizumab,Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Vobarilizumab,Volociximab, Vorsetuzumab mafodotin, Votumumab, Xentuzumab, Zalutumumab,Zanolimumab, Zatuximab, Ziralimumab, or Zolimomab aritox.

In some embodiments, the therapeutic is an immunotherapy. In someembodiments, the immunotherapy is a PD-1 inhibitor such as a PD-1antibody, a PD-L1 inhibitor such as a PD-L1 antibody, a CTLA-4 inhibitorsuch as a CTLA-4 antibody, a CSF-1 R inhibitor, an IDO inhibitor, an A1adenosine inhibitor, an A2A adenosine inhibitor, an A2B adenosineinhibitor, an A3A adenosine inhibitor, an arginase inhibitor, or an HDACinhibitor. In some embodiments, the immunotherapy is a PD-1 inhibitor(e.g., nivolumab, pembrolizumab, pidilizumab, BMS 936559, andMPDL328OA). In some embodiments, the immunotherapy is a PD-L1 inhibitor(e.g., atezolizumab and MED14736). In some embodiments, theimmunotherapy is a CTLA-4 inhibitor (e.g., ipilimumab). In someembodiments, the immunotherapy is a CSF-1 R inhibitor (e.g.,pexidartinib and AZD6495). In some embodiments, the immunotherapy is anIDO inhibitor (e.g., norharmane, rosmarinic acid, andalpha-methyl-tryptophan). In some embodiments, the immunotherapy is anA1 adenosine inhibitor (e.g., 8-cyclopentyl-1,3-dimethylxanthine,8-cyclopentyl-1,3-dipropylxanthine, 8-phenyl-1,3-dipropylxanthine,bamifylline, BG-9719, BG-9928, FK-453, FK-838, rolofylline, or N-0861).In some embodiments, the immunotherapy is an A2A adenosine inhibitor(e.g., ATL-4444, istradefylline, MSX-3, preladenant, SCH-58261,SCH-412,348, SCH-442,416, ST-1535, VER-6623, VER-6947, VER-7835,viadenant, or ZM-241,385). In some embodiments, the immunotherapy is anA2B adenosine inhibitor (e.g., ATL-801, CVT-6883, MRS-1706, MRS-1754,OSIP-339,391, PSB-603, PSB-0788, or PSB-1115). In some embodiments, theimmunotherapy is an A3A adenosine inhibitor (e.g., KF-26777, MRS-545,MRS-1191, MRS-1220, MRS-1334, MRS-1523, MRS-3777, MRE-3005-F20,MRE-3008-F20, PSB-11, OT-7999, VUF-5574, and SSR161421). In someembodiments, the immunotherapy is an arginase inhibitor (e.g., anarginase antibody, (2s)-(+)-amino-5-iodoacetamidopentanoic acid,NG-hydroxy-L-arginine, (2S)-(+)-amino-6-iodoacetamidohexanoic acid, or(R)-2-amino-6-borono-2-(2-(piperidin-1-yl)ethyl)hexanoic acid. In someembodiments, the immunotherapy is an HDAC inhibitor (e.g., valproicacid, SAHA, or romidepsin).

In some embodiments, the therapeutic can be ledipasvir/sofosbuvir,insulin glargine, lenalidomide, pneumococcal 13-valent conjugatevaccine, fluticasone/salmeterol,elvitegravir/cobicistat/emtricitabine/tenofovir alafenamide,emtricitabine, rilpivirine and tenofovir alafenamide,emtricitabine/tenofovir alafenamide, grazoprevir/elbasvir, coagulationfactor VIIa recombinant, epoetin alfa, Aflibercept or etanercept.

In some embodiments, the therapeutic or diagnostic agents is Abatacept,AbobotulinumtoxinA, Agalsidase beta, Albiglutide, Aldesleukin,Alglucosidase alfa, Alteplase (cathflo activase), Anakinra, Asfotasealfa, Asparaginase, Asparaginase erwinia chrysanthemi, Becaplermin,Belatacept, Collagenase, Collagenase clostridium histolyticum,Darbepoetin alfa, Denileukin diftitox, Dornase alfa, Dulaglutide,Ecallantide, Elosulfase alfa, Etanercept-szzs, Filgrastim,Filgrastim-sndz, Galsulfase, Glucarpidase, Idursulfase,IncobotulinumtoxinA, Interferon alfa-2b, Interferon alfa-n3, Interferonbeta-1 a, Interferon beta-1 b, Interferon gamma-1b, Laronidase, Methoxypolyethylene glycol-epoetin beta, Metreleptin, Ocriplasmin,OnabotulinumtoxinA, Oprelvekin, Palifermin, Parathyroid hormone,Pegaspargase, Pegfilgrastim, Peginterferon alfa-2a, Peginterferonalfa-2a co-packaged with ribavirin, Peginterferon alfa-2b, Peginterferonbeta-1a, Pegloticase, Rasburicase, Reteplase, Rilonacept,RimabotulinumtoxinB, Romiplostim, Sargramostim, Sebelipase alfa,Tbo-filgrastim, Tenecteplase, or Ziv-aflibercept.

In some embodiments, the diagnostic agent is tuberculin purified proteinderivative, hyrotropin alpha, secretin, soluble transferrin receptor,troponin, B-type natriuretic peptide, iobenguane I 123, florbetapir F18, perflutren, gadoterate meglumine, florbetaben F 18, flutemetamol F18, gadoterate meglumine, isosulfan blue, regadenoson, technetium Tc 99mtilmanocept, florbetaben F 18, perflutren, regadenoson, or flutemetamolF 18.

Formulations

The electrosprayed particles can be suspended in a non-aqueous oraqueous liquid or gel (or a mixture thereof) to form a suspensionformulation. The non-aqueous liquid can be an organic solvent or anionic liquid or some combination thereof. In some embodiments, theorganic solvent is benzyl alcohol, benzyl benzoate, coconut oil,cottonseed oil, fish oil, grape seed oil, hazelnut oil, hydrogenatedvegetable oils, olive oil, palm seed oil, peanut oil, peppermint oil,safflower oil, sesame oil, soybean oil, sunflower oil, walnut oil,acetone, ethyl acetate, ethyl lactate, dimethylacetamide, dimethylisosorbide, dimethyl sulfoxide, glycofurol, diglyme, methyl tert-butylether, N-methyl pyrrolidone, perfluorodecalin, polyethylene glycol,2-pyrrolidone, tetrahydrofurfuryl alcohol, trigylcerides, triglyceridesof the fractionated plant fatty acids C8 and C10 (e.g., MIGLYOL® 810 andMIGLOYL® 812N), propylene glycol diesters of saturated plant fatty acidsC8 and C10 (e.g., MIGLYOL® 840), ethyl oleate, ethyl caprate, dibutyladipate, fatty acid esters, hexanoic acid, octanoic acid, triacetin,diethyl glycol monoether, gamma-butyrolactone, eugenol, clove bud oil,citral, limonene, and any combination thereof. In some embodiments, theionic liquid includes pyridinium, pyridazinium, pyrimidinium,pyrazinium, imidazolium, pyrazolium, thiazolium, oxazolium, triazolium,ammonium, sulfonium, halides, sulfates, sulfonates, carbonates,phosphates, bicarbonates, nitrates, acetates, PF₆—, BF₄—, triflate,nonaflate, bis(trifyl)amide, trifluoroacetate, heptafluorobutanoate,haloaluminate, or any combination thereof. Aqueous liquids forsuspension include water, 0.9% saline, lactated Ringer's solution,dextrose 5% or a buffer. The buffer may include, e.g., acetate buffer,histidine buffer, succinate buffer, HEPES buffer, tris buffer, carbonatebuffer, citrate buffer, phosphate buffer, glycine buffer, barbitalbuffer, and cacodylate buffer. The medium for suspension may furtherinclude another component, such as, e.g., a carbohydrate, a pH adjustingagent, a salt, a chelator, a mineral, a polymer, a surfactant, a proteinstabilizer, an emulsifier, an antiseptic, an amino acid, an antioxidant,a protein, an organic solvent, or nutrient media. In some embodiments,each of the other components is, independently, at 0.0001 to 99% (w/v)of the medium, e.g., at 0.0001 to 90% (w/v), at 0.0001 to 50% (w/v), at0.0001 to 10% (w/v), at 0.0001 to 1% (w/v), or at 0.0001 to 0.1% (w/v).One of ordinary skill in the art would be able to determine anappropriate amount of the other components in the medium. Carbohydratesdextran, trehalose, sucrose, agarose, mannitol, lactose, sorbitol, ormaltose. The pH adjusting agent may be, e.g., acetate, citrate,glutamate, glycinate, histidine, lactate, maleate, phosphate, succinate,tartrate, bicarbonate, aluminum hydroxide, phosphoric acid, hydrochloricacid, DL-lactic/glycolic acids, phosphorylethanolamine, tromethamine,imidazole, glyclyglycine, or monosodium glutamate. Exemplary salts aresodium chloride, calcium chloride, potassium chloride, sodium hydroxide,stannous chloride, magnesium sulfate, sodium glucoheptonate, sodiumpertechnetate, or guanidine hydrochloride. The chelator can be, e.g.,disodium edetate. The mineral can be, e.g., calcium, zinc, or titaniumdioxide. Examples of polymers are propyleneglycol, glucose star polymer,silicone polymer, polydimethylsiloxane, polyethylene glycol,carboxymethylcellulose, poly(glycolic acid), poly(lactic-co-glycolicacid), or polylactic acid. The surfactant may be, e.g., polysorbate,magnesium stearate, sodium dodecyl sulfate, Triton N-101, glycerin, orpolyoxyethylated castor oil. Protein stabilizers includeacetyltryptophanate, caprylate, or N-acetyltryptophan. The emulsifiercan be, e.g., polysorbate 80, polysorbate 20, sorbitan monooleate,ethanolamine, polyoxyl 35 castor oil, poloxyl 40 hydrogenated castoroil, carbomer 1342, a corn oil-mono-di-triglyceride, a polyoxyethylatedoleic glyceride, or a poloxamer. Exemplary antiseptics include phenol,m-cresol, benzyl alcohol, 2-phenyloxyethanol, chlorobutanol, neomycin,benzethonium chloride, gluteraldehyde, or beta-propiolactone. The aminoacid may be alanine, aspartic acid, cysteine, isoleucine, glutamic acid,leucine, methionine, phenylalanine, pyrrolysine, serine, selenocysteine,threonine, tryptophan, tyrosine, valine, asparagine, L-arginine,histidine, glycine, or glutamine, e.g., asparagine, L-arginine,histidine, glycine, or glutamine. The antioxidant can be glutathione,ascorbic acid, cysteine, or tocopherol. The protein can be protamine,protamine sulfate, or gelatin.

For aqueous suspension formulations, high concentration trehalosesolutions can stabilize the particles in suspension and preventpremature dissolution. The sugar acts as a “crowder” molecule, i.e., acrowding agent, that occupies a large volume, increasing short rangeprotein-protein attractive interactions in some embodiments. Thisstabilizing effect has also been described for other crowding agents inwater such as polymers, e.g., PEG 300, and organic molecules, e.g.,N-methyl-2-pyrrolidone (Miller et al. J. Pharm. Sci., 2012, 101,3763-3778).

The viscosity of the suspension can range from 0.27 to 200 cP, e.g.,0.27 to 50 cP, 1 to 30 cP, or 20 to 50 cP. In some embodiments, theviscosity of the suspension ranges from 0.27 to 200 cP, e.g., 0.27 to100 cP, 0.27 to 50 cP, 0.27 to 30 cP, 1 to 20 cP, or 1 to 15 cP. Incertain embodiments, the viscosity is measured at a shear rate in theNewtonian regime. In other embodiments, the viscosity is measured at ashear rate of 100 s⁻¹ or greater, e.g., at 1000 s⁻¹ or greater than 1000s⁻¹. The suspension may include from 5 to 90% particles by volume, e.g.,e.g., 20 to 90%, 40 to 80%, 50 to 60%, or 70 to 90%. The suspension mayhave a concentration of the first therapeutic or diagnostic agent from0.0001 to 1000 mg/mL, e.g., from 100 to 900, 150 to 800, or 200 to 700mg/mL.

In some embodiments of the invention described herein, highconcentrations of therapeutic or diagnostic agents in the particles andhigh concentrations of particles in the non-aqueous or aqueous liquidare possible. The latter may be achieved by mixing particles of varioussizes.

In some embodiments, one or more therapeutic or diagnostic agents can bein the particles and outside of the particles, i.e., in the non-aqueousor aqueous liquid. The therapeutic or diagnostic agent included in thenon-aqueous or aqueous liquid can be the same or different than thatemployed in the particle. The one or more therapeutic or diagnosticagent can reduce pain or inflammation during administration. Theconcentation of the therapeutic agent in the pharmaceutical compositionoutside of the particles can range, e.g., from 0.0001 to 1000 mg/mL.

In some embodiments, the particles are sprayed and collected for use ina needle-free injector or in an inhalation or other nasal deliverysystem. In some embodiments, the particles are stored as a dry powder.In some embodiments, the dry powder is reconstituted shortly beforeadministration of a formulation in which the therapeutic or diagnosticagents are dissolved in an aqueous or non-aqueous solution. Such aparadigm is beneficial in some cases for circumventing the relativeinstability or degradation of certain therapeutic or diagnostic agentsstored in an aqueous or non-aqueous solution form, rather than a drypowder form.

Needle-free injection systems may include liquids, suspensions, powdersor projectiles. Powders are suitable for long term storage and can beinjected at home without prior reformulation and preparation using suchsystems. Powders require an injection chamber filled with solid drug anda nozzle for firing particle into the skin. In brief, in someembodiments, the particles exit the nozzle with a gas stream and impingethe skin surface. This leads to small perforations or holes in which theparticles are deposited. They penetrate the stratum corneum before beingdistributed completely into the stratum corneum and viable epidermis.Particles for needle-free injection may have a density of around 1 g/cm³or higher and a mean diameter greater than 20 μm.

In some embodiments, the particles are administered via a dual-chambersyringe device such as LyoTwist. The particles exist as a dry powder inone chamber of the device while the second chamber is occupied by anaqueous or non-aqueous carrier liquid. The components of the twochambers are mixed briefly before administration.

Methods of Use

The pharmaceutical compositions including suspensions or dry forms ofthe invention may be administered in a suitable dosage that may beadjusted as required, depending on the clinical response. Compositionsmay also be used cosmetically. The dosage of the pharmaceuticalcomposition can vary depending on many factors, such as thepharmacokinetics of the therapeutic or diagnostic agents; the mode ofadministration; the age, health, and weight of the recipient; the natureand extent of the symptoms; the frequency of the treatment, and the typeof concurrent treatment, if any; and the clearance rate of thetherapeutic or diagnostic agents in the animal to be treated. One ofskill in the art can determine the appropriate dosage based on the abovefactors. Administration may occur daily, weekly, every two weeks, everythree weeks, monthly, or any other suitable interval. In general,satisfactory results may be obtained when the therapeutic or diagnosticagent is administered to a human at a dosage of, for example, between0.01 mg/kg and 70 mg/kg (measured as the solid form). In someembodiments, the dosage may range from 0.01 mg/kg to 1 mg/kg. Doseranges include, for example, between 30 mg and 5000 mg. In someembodiments, at least 30, 100, 500, 1000, 2000, or 5000 mg of thecompound is administered. Preferred dose ranges include, for example,between 1-30 mg/kg, such as 1-10 mg/kg.

The volume delivered will depend on the indication and the route ofdelivery. In one embodiment, a dose of more than 0.5 mL, e.g., more than2 mL, of a pharmaceutical composition having a viscosity less than 50cP, e.g., less than 30 cP, is administered, e.g., injected into skin ofan animal. In some embodiments, a dose of more than 1.5 mL, e.g., morethan 5 mL, of a pharmaceutical composition having a viscosity less than50 cP, e.g., less than 30 cP, is administered, e.g., injected into skinof an animal.

The pharmaceutical composition may be administered by any suitablemethod, for example, by auricular, buccal, conjunctival, cutaneous,dental, electro-osmotical, endocervical, endosinusial, endotracheal,enteral, epidural, extra-amniotical, extracorporeal, infiltration,interstitial, intra-abdominal, intra-amniotical, intra-arterial,intra-articular, intrabiliary, intrabronchial, intrabursal,intracardial, intracartilaginous, intracaudal, intracavernous,intracavitary, intracerebral, intracisternal, intracorneal,intracoronal, intracoronary, intracorporus cavernosum, intradermal,intradiscal, intraductal, intraduodenal, intradural, intraepidermal,intraesophageal, intragastrical, intragingival, intraileal,intralesional, intraluminal, intralymphatical, intramedullar,intrameningeal, intramuscular, intraocular, intraovarian,intrapericardial, intraperitoneal, intrapleural, intraprostatical,intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous,intratesticular, intrathecal, intrathoracic, intratubular, intratumor,intratympanic, intrauterine, intravascular, intravenous, intravenousbolus, intravenous drip, intraventricular, intravesical, intravitreal,iontophoresis, irrigation, laryngeal, nasal, nasogastrical, occlusivedressing technique, ophthalmical, oral, oropharyngeal, parenteral,percutaneous, periarticular, peridural, perineural, periodontal, rectal,inhalation, retrobulbar, soft tissue, subarachnoidial, subconjunctival,subcutaneous, sublingual, submucosal, topical, transdermal,transmucosal, transplacental, transtracheal, transtympanic, ureteral,urethral, or vaginal administration and the pharmaceutical compositionsformulated accordingly.

EXAMPLES

The methods disclosed herein have been utilized in separate instances toproduce particles including bovine serum albumin (BSA), particlesincluding whole human IgG, and particles including one of three discretemonoclonal antibodies. Various analytical techniques have been appliedto assess the physical characteristics of the particles themselves andthe structural and functional properties of the proteins they include.Scanning electron microscopy and associated image analysis have beenused to study the particle morphology and size distribution,respectively. Smooth particles of high sphericity were achieved forcertain excipient profiles while facile control of the mean size waspossible over a broad range with low dispersity. In certain instances,the particle surfaces were decorated with excipients. Density and watercontent measurement demonstrated that the particles approachedcrystalline packing efficiencies and retained very low levels ofresidual moisture, particularly in instances in which secondarydesiccation was employed. The functional properties of the proteins werealso preserved, as evidenced by ELISA and binding assays performed onreconstituted particles. This was corroborated by size exclusion HPLCanalysis indicating that the process had a minimal or even remedialeffect on the degree of inter-protein association. Finally, suspensionof the particles in various carrier media showed that ultra-highloadings, e.g., >300 mg/mL, were possible, and the apparent viscosity ofthe particle-medium complex was below 20 cP.

Materials and Methods

Bovine serum albumin and human IgG were obtained from Sigma-Aldrich(A2934, >98%) and Innovative Research (IRHUGGF-LY, >97%), respectively,as lyophilized powders. Biosimilar versions of three marketed monoclonalantibody products (mAb1, mAb2, mAb3) were obtained in aqueous solution.The formulations were congruent with the corresponding FDA label.Concentration columns were procured from Millipore Sigma (Amicon® Ultra15 mL Filters for Protein Purification and Concentration with a 3 kDacut off) and used where necessary to reach the desired proteinconcentration for the electrospray feed solution. All excipients and thecarrier medium, benzyl benzoate, were purchased from Sigma-Aldrich andused as received.

Electrospray Particle Formation

Unless otherwise noted, a conventional electrospray apparatus of customdesign was used to prepare the particle samples disclosed herein. Thisapparatus comprised a 30-gauge blunt disposable syringe needle(89134-196, VWR International) attached to a Harvard Apparatus Model 33dual-channel syringe pump. The needle was charged by a MatsusadaEQ-30P1-LCtG high voltage DC power supply. For select samples, theneedle was replaced by a Sono-Tek 120 kHz ultrasonic atomizer nozzledriven at a power of 4.5 W.

Lyophilization

The particles for lyophilized samples, i.e., samples marked as havinggone through secondary desiccation, were loaded into microcentrifugetubes and subjected to snap freezing by immersion in liquid nitrogen forapproximately 10 min. The samples were then loosely covered andtransferred to a Labconoco Freezone lyophilizer for approximately 48hours at a pressure of approximately 0.035 Torr.

Scanning Electron Microscopy

Electron micrographs were collected for select samples with a HitachiTM3030Plus tabletop microscope. The samples were immobilized onconductive tape and examined in a low-vacuum anti-charging environment,obviating the need for sample preparation.

Optical Microscopy

Select samples were prepared for imaging by vortexing 5 mg of particleswith benzyl benzoate (20 microliters). Samples were then pipetted onto aglass slide and imaged using a Celena microscope by Logos Biosystems.

Image Analysis

Select microscopy images were chosen for further analysis on the basisof (i) minimal particle overlapping, (ii) good contrast between theparticles and the background, and (iii) a resolution providing forparticle occupancies of at least 10 pixels. This allowed for particlesto be easily identified and reduced resolution-based error. A binarythreshold was applied to separate the particles from background, and awatershed segmentation algorithm was applied to ensure that individualparticles were measured separately. The ImageJ tool “Analyze Particles”was then applied on the binary picture with the following parameters:circularity between 0.5 and 1.0; size between 5 and infinity squaremicrons; exclude on edges; fill holes. The outlines of the identifiedparticles were overlaid onto the original image. Particles which weremisidentified, such as clusters that were identified as a singleparticle or particles whose outlines do not match the particle, werethen discarded. Missing particles were measured by manually tracing theparticle's outline and using ImageJ's Measure tool.

Density Analysis

The skeletal density of electrosprayed particles from select samples wasdetermined by examining approximately 0.1 g of powder with an AccuPyc111340 gas displacement pycniometry system.

Water Content Analysis

The residual moisture in electrosprayed particles from select sampleswas determined by placing approximately 0.1 g of powder in a vacuum ovenwith a Karl Fischer titrator and heating the sample.

Salt Content Analysis

The salt content of electrosprayed particles from select samples wasdetermined by elemental analysis for chlorine. This was performed byflask combustion followed by ion chromatography.

Zeta Potential Analysis

Zeta potential analysis was carried out on select samples using aMalvern Instruments ZetaSizer Nano. ZS. Solutions were made up at 8mg/mL in DI water and placed in a Malvern Instruments INC DTS1070 foldedcapillary cell.

ELISA Assay

ELISA assay was used on select samples to detect human antibody in adenaturation sensitive manner. Human IgG was first plated in PBS for 1hour, followed by washing with wash buffer (PBS+0.05% Tween20) threetimes for 4 minutes, followed by blocking with 2% BSA (Sigma) in washbuffer for 45 minutes, followed incubation with dilute (20 μg/ml)protein A-HRP (Abcam) for 45 minutes, followed by wash buffer threetimes for 3 minutes, followed by incubation with TMB (Abcam) for 10minutes, finally followed by quenching of the reaction with STOPsolution (Abcam). The colorimetric readout was conducted on a ThermoMultiskan Spectrum.

Monoclonal Antibody Binding Assay

Monoclonal antibodies from select samples were assessed for cellularbinding ability utilizing cells that express the appropriate cellsurface receptors. Cells were incubated for 30 minutes at 4° C. withmonoclonal antibodies at respective concentrations and then spun down at2000 rpm followed by washing with PBS three times. Cells were thenincubated with secondary goat anti-human Fab antibody fluorescentlylabeled with PE for 30 minutes at 4° C. The cells were then spun down at2000 rpm followed by washing with PBS three times. The cells were thenre-suspended and then analyzed on an Attune Flow Cytometer (Invitrogen).

Size Exclusion Chromatography

The quantification of size variants in select samples was determined bysize exclusion chromatography. This analysis utilized Advanced BioSEC),column, 7.8 mm ID×30 cm, 3 μm (Agilent) run on an HPLC system (1100,Agilent). The mobile phases were 0.2 M potassium phosphate and 0.25 Mpotassium chloride at pH 6.0. The chromatography was run isocracticallyat a flow rate of 1.0 mL/min for 15 minutes. The column temperature wasmaintained at ambient 25° C. and the eluent absorbance was monitored at280 nm. Each monoclonal antibody was diluted with its respectiveformulation buffer to 1 mg/mL. Their injection volume was 20 μL.

Suspension Preparation

For select samples, particles were weighed in a 2-mL EppendorfMicrocentrifuge tube. Based on the measured powder density, theappropriate amount of suspension vehicle was added to prepare asuspension of the desired concentration. Samples were then vortexed for30 seconds.

Viscosity Measurement

The viscosities of solutions and suspensions were measured through theuse of an AR-G2 rheometer (TA Instruments) with a cone and plategeometry (20 mm/2°). The samples were measured every 30 seconds for 5minutes at a shear rate of 1000 per second. In certain instances, theintrinsic viscosity factor [η] was calculated on the basis of themeasurements. This was an indication of the extent to which particlescontributed to the viscosity of the suspension. It was computed from theKrieger-Dougherty equation:

$\eta_{rel} = \left( {1 - \frac{\varphi}{\varphi_{m}}} \right)^{{- {\lbrack\eta\rbrack}}\varphi_{m}}$

where η_(rel) is the apparent viscosity of the suspension normalized bythe viscosity of the suspension medium, ϕ is the volume fraction ofparticles in suspension, and ϕ_(m) is the maximum feasible volumefraction for the particles (approximately 0.6 in theory andapproximately 0.5 in practice (M. A. Miller, J. D. Engstrom, B. S.Ludher, K. P. Johnston, Langmuir, 2010, 26, 1067)). The value of [η] wastypically above the limiting value of 2.5 dL/g, the so-called Einsteinvalue, depending on the effects of particle shape, electroviscosity, andsolvation.

Results and Discussion Particle Size, Shape, and Chemical Composition

4 mL of BSA solution was prepared in deionized water at a concentrationof about 80 mg/mL. The solution was electrosprayed with a flow rate of0.4 mL/hr and an applied voltage of about 13.3 kV. After primarydesiccation and optical microscopy, ImageJ analysis indicated a meanparticle size of 19.95 μm with a dispersity index of 0.27 (FIG. 11).

3 mL of desalted IgG solution was prepared at a concentration of 50mg/mL by exchanging PBS buffer solution with deionized water through theuse of a concentration column. The solution was electrosprayed with aflow rate of 0.4 mL/hr and an applied voltage of 10.6 kV. After primarydesiccation and SEM imaging, ImageJ indicated a mean particle size of18.06 μm with a dispersity index of 0.12 (FIG. 12). Salt content wasfound to be less than 1.5 wt % (note that the initial PBS buffercontained NaCl at around 8 g/L). The particle density and water contentafter primary desiccation were 1.324 g/mL and 7-10 wt %, respectively. Aresidual moisture content of less than 3 wt % was achieved afterimplementation of a secondary desiccation step.

5 mL of desalted mAb solution was prepared at a concentration of about20 mg/mL by exchanging a buffer solution with deionized water in aconcentration column. The solution was electrosprayed with a flow rateof about 0.4 mL/hr and an applied voltage of about 11.8 kV. Afterprimary drying and optical microscopy, ImageJ analysis indicated a meansize of 16.02 μm with a dispersity index of 0.09 (FIG. 13).

Aggregate Analysis

1 mL of a solution of mAb1 at a concentration of 20 mg/mL with anexcipient at a concentration of 8 mg/mL was prepared at pH 6.5 (feedsolution). The solution was electrosprayed at a flow rate of 0.4 mL/hrwith an applied voltage of 11.9 kV. Aggregate analysis was performed byHPLC after (i) primary desiccation, (ii) secondary desiccation, (iii)and storage at accelerated conditions (7 days, 40° C.). The sizedistribution of aggregates was compared to those observed in both thefeed solution and the label formulation. After seven days of acceleratedstorage, the particle formulation had about 0.2% fewer aggregates thanthe label formulation when stored under similar conditions (see Table 2below).

TABLE 2 Formulation % Monomers % Aggregates Label 96.8 3.2 Feed 96.6 3.4Electrosprayed t = 0 w/ 97.3 2.7 primary desiccation Electrosprayed t =0 days w/ 96.6 3.4 secondary desiccation Label t = 7 days 95.7 4.3Electrosprayed t = 7 days 95.9 4.1

IgG Function by ELISA Assay

A solution of IgG at a concentration of 50 mg/mL was electrosprayed at aflow rate of about 0.4 mL/hr with an applied voltage of 10.6 kV.Particles were reconstituted in PBS after primary desiccation so thatthe IgG contained therein could be analyzed by ELISA assay. FIG. 15shows that the signal for the reconstituted IgG was equivalent to thatof the control sample in FIG. 14, indicating that the binding activityfor the Fc (constant) domain of the electrosprayed protein waspreserved.

Cellular Binding Activity (In Vitro Indicator for Therapeutic Activity)for mAb1 and mAb2

A solution of mAb1 at a concentration of 20 mg/mL was electrosprayed ata flow rate of about 0.4 mL/hr with an applied voltage of 11.8 kV. Asecond solution of mAb2 at a concentration of 20 mg/mL waselectrosprayed at a flow rate of about 0.4 mL/hr with an applied voltageof 11.9 kV. The particles were subjected to primary desiccation. In FIG.16 and FIG. 17, the binding activities of the label formulations formAb1 and mAb2 are shown, as assessed by flow cytometry. Thermaldenaturation controls and a negative mAb control with no affinity forthe target expressed by the cell line indicated that the assay wassensitive to thermal denaturation and selectivity. Particles includingmAb1 (FIG. 16) and particles including mAb2 (FIG. 17) were thenreconstituted in PBS and assayed for binding activity. The resultsindicated that full binding activity of the antibodies was preservedthrough the electrospray particle formation process.

In FIG. 18, the thermal stability of mAb1 particles was assessed over aseven-day period of accelerated storage at 40° C. The particles weresplit into two samples, one stored as a dry powder and the other storedin benzyl benzoate. The particles were reconstituted in PBS afterstorage, at which point the flow cytometry assay again indicatedpreservation of the cellular binding activity as compared to unprocessedmaterial (label formulation) without storage.

Viscosity of an IgG Particle Suspension

Particles of desalted IgG and mAb1 were prepared by electrospray asdescribed above. These particles were individually suspended in benzylbenzoate and analyzed on a rheometer at a shear rate of approximately1000 per second. Both suspensions exhibited similar rheological behaviorand substantially outperformed an aqueous formulation of mAb1 (FIG. 19).Suspension formulations of greater than 300 mg/mL of either agent wereobserved to have an apparent viscosity below approximately 20 cP.Moreover, IgG particles in suspension at 500 mg/mL were found to have an[η] value of about 2.64 dL/g, indicating that the viscosity wasprimarily governed by excluded volume effects. This is to be comparedagainst aqueous solutions of various proteins in which intermolecularinteractions can significantly contribute to the apparent viscosity ofthe medium, e.g., aqueous IgG is typified by [η] values near 6.5 dL/g(E. Longman, S. E. Harding, N. Mareineke, LCGC North America, 2006, 24,1, 64-72).

Comparison of Human IgG Particles Produced With and Without an ElectricField

Particles of human IgG were produced by drying the drops of an aerosolof aqueous IgG (approximately 16 mg/mL IgG, 4 mg/mL NaCI) which wasgenerated by an ultrasonic nozzle. The aerosol formation for one groupof particles (Group A) was assisted by an electric field (FIG. 20),applied by enforcing an 8 kV bias between the nozzle and an electrodestationed several centimeters downstream, and for another group it wasnot (Group B) (FIG. 21).

The mean sizes of the particles in Groups A and B were 6.5 and 10.1 μm,respectively, indicating that the electric field acted to reduce theaverage drop size. The standard deviations were 1.8 and 3.4 μm,respectively. The particles were then reconstituted in water andanalyzed via HPLC. Aggregates were found to be 7.9% (Group A) and 6.9%(Group B) of the protein populations, as compared to 14.1% aggregates inthe feed solution. The zeta potentials for the same reconstitutedmaterial were 3.95 mV (Group A) and 6.91 mV (Group B).

Surface Prominent excipients in Human IgG Particles Produced byElectrospray

A solution of human IgG at about 80 mg/mL and a salt at about 30 mg/mLwas electrosprayed at a flow rate of 0.4 mL/hr with an applied voltageof 12.2 kV. Particles were produced after primary desiccation of thedrops and visualized with SEM (FIG. 22, FIG. 23). Crystalline orsemi-crystalline salt was observed at the surface of the particles inlarge quantities.

A solution of human IgG at about 80 mg/mL, a salt at about 20 mg/mL, anda sugar at about 10 mg/mL was electrosprayed at a flow rate of 0.4 mL/hrwith an applied voltage of 12 kV. Particles were produced after primarydesiccation of the drops and visualized with SEM (FIG. 24, FIG. 25). Thesalt and/or sugar was observed at the surface of the particles in largequantities.

A solution of monoclonal antibody comprising mAb3 at about 70 mg/mL anda sugar at about 46 mg/mL was electrosprayed at a flow rate of 0.4 mL/hrwith an applied voltage of 12.1 kV. Particles were produced afterprimary desiccation of the drops and visualized with SEM (FIG. 26, FIG.27). The sugar was observed at the surface of the particles in largequantities.

Other embodiments are in the claims.

1. A method of electrospraying a first liquid comprising a firsttherapeutic or diagnostic agent to form droplets and evaporating thefirst liquid to produce particles from the droplets, wherein thetherapeutic or diagnostic agent in the particles has 0.5 to 1.0 activityper unit.
 2. The method of claim 1, wherein the concentration of thefirst therapeutic or diagnostic agent in the first liquid is from 0.0001to 1000 mg/mL.
 3. The method of claim 1, wherein the first liquid isaqueous.
 4. The method of claim 3, wherein the aqueous first liquid isselected from the group consisting of water, 0.9% saline, lactatedRinger's solution, dextrose 5%, or a buffer.
 5. (canceled)
 6. The methodof claim 1, wherein the first liquid further comprises a carbohydrate, apH adjusting agent, a salt, a chelator, a mineral, a polymer, asurfactant, a protein stabilizer, an emulsifier, an antiseptic, an aminoacid, an antioxidant, a protein, an organic solvent, or a nutrientmedium.
 7. The method of claim 6, wherein the carbohydrate is dextran,trehalose, sucrose, agarose, mannitol, lactose, sorbitol, or maltose. 8.(canceled)
 9. The method of claim 6, wherein the salt is sodiumchloride, calcium chloride, potassium chloride, sodium hydroxide,stannous chloride, magnesium sulfate, sodium glucoheptonate, sodiumpertechnetate, or guanidine hydrochloride. 10-14. (canceled)
 15. Themethod of claim 6, wherein the emulsifier is polysorbate 80, polysorbate20, sorbitan monooleate, ethanolamine, polyoxyl 35 castor oil, polyoxyl40 hydrogenated castor oil, carbomer 1342, a cornoil-mono-di-triglyceride, a polyoxyethylated oleic glyceride, or apoloxamer. 16-24. (canceled)
 25. The method of claim 1, wherein theparticles have diameters from 0.1 to 1000 μm.
 26. The method of claim 1,wherein the particles have a polydispersity index from 0.05 to 0.9.27-34. (canceled)
 35. The method of claim 1, wherein the firsttherapeutic or diagnostic agent is selected from the group consisting ofnucleic acids, antibodies, peptides, proteins, cells, carbohydrates,chemical drugs, contrast agents, magnetic particles, polymer beads,metal nanoparticles, metal microparticles, quantum dots, antioxidants,antibiotic agents, hormones, nucleoproteins, polysaccharides,glycoproteins, lipoproteins, steroids, analgesics, local anesthetics,anti-inflammatory agents, anti-microbial agents, chemotherapeuticagents, exosomes, outer membrane vesicles, vaccines, viruses,bacteriophages, adjuvants, vitamins, minerals, organelles, and anycombination thereof. 36-152. (canceled)
 153. A method of formingparticles by electrospraying an annular stream of an encapsulant towarda collector, and centrally with respect to the annular stream of theencapsulant, electrospraying a stream of a liquid comprising a firsttherapeutic or diagnostic agent toward the collector, the particlesbeing collected on the collector, the concentration of the firsttherapeutic or diagnostic agent in the liquid ranging from 1 to 1000mg/mL. 154-157. (canceled)
 158. The method of claim 153, wherein theliquid is aqueous. 159-160. (canceled)
 161. The method of claim 153,wherein the liquid further comprises a carbohydrate, a pH adjustingagent, a salt, a chelator, a mineral, a polymer, a surfactant, a proteinstabilizer, an emulsifier, an antiseptic, an amino acid, an antioxidant,a protein, an organic solvent, or a nutrient medium.
 162. The method ofclaim 161, wherein the carbohydrate is dextran, trehalose, sucrose,agarose, mannitol, lactose, sorbitol, or maltose.
 163. (canceled) 164.The method of claim 161, wherein the salt is sodium chloride, calciumchloride, potassium chloride, sodium hydroxide, stannous chloride,magnesium sulfate, sodium glucoheptonate, sodium pertechnetate, orguanidine hydrochloride. 165-169. (canceled)
 170. The method of claim161, wherein the emulsifier is polysorbate 80, polysorbate 20, sorbitanmonooleate, ethanolamine, polyoxyl 35 castor oil, polyoxyl 40hydrogenated castor oil, carbomer 1342, a corn oil-mono-di-triglyceride,a polyoxyethylated oleic glyceride, or a poloxamer. 171-182. (canceled)183. The method of claim 153, wherein the first therapeutic ordiagnostic agent is selected from the group consisting of nucleic acids,antibodies, peptides, proteins, cells, carbohydrates, chemical drugs,contrast agents, magnetic particles, polymer beads, metal nanoparticles,metal microparticles, quantum dots, antioxidants, antibiotic agents,hormones, nucleoproteins, polysaccharides, glycoproteins, lipoproteins,steroids, analgesics, local anesthetics, anti-inflammatory agents,anti-microbial agents, chemotherapeutic agents, exosomes, outer membranevesicles, vaccines, viruses, bacteriophages, adjuvants, vitamins,minerals, organelles, and any combination thereof. 184-214. (canceled)215. The method of claim 153, wherein the particles have diameters from0.1 to 1000 μm.
 216. The method of claim 153, wherein the particles havea polydispersity index from 0.05 to 0.9. 217-298. (canceled)